Genetic control of flowering

ABSTRACT

A flowering characteristic of a plant, particularly the timing of flowering, is controlled by the expression of the Late Elongated Hypocotyl (LHY) gene of  Arabidopsis thaliana  or a mutant, variant, allele, derivative, or homologue thereof. Over-expression may be used to delay flowering in a transgenic plant. The promoter of the gene regulates transcription in accordance with the circadian rhythm and may be used to control expression of genes whose products are only required or desired at certain times of the day.

This invention relates to the genetic control of flowering in plants andthe cloning and expression of genes involved therein. More particularly,the invention relates to the cloning and expression of the LateElongated Hypocotyl (LHY) gene of Arabidopsis thaliana, and homologuesfrom other species, and manipulation and use of the gene in plants.

BACKGROUND OF THE INVENTION

Efficient flowering in plants is important, particularly when theintended product is the flower or the seed produced therefrom. Oneaspect of this is the timing of flowering: advancing or retarding theonset of flowering can be useful to farmers and seed producers. Anunderstanding of the genetic mechanisms which influence floweringprovides a means for altering the flowering characteristics of thetarget plant. Species for which flowering is important to cropproduction are numerous, all crops which are grown from seed, withimportant examples being the cereals, rice and maize, probably the mostagronomically important in warmer climatic zones, and wheat, barley,oats and rye in more temperate climates. Important seed products are oilseed rape and canola, sugar beet, maize, sunflower, soybean and sorghum.Many crops which are harvested for their roots or leaves are, of course,grown annually from seed and the production of seed of any kind is verydependent upon the ability of the plant to flower, to be pollinated andto set seed. Delaying flowering is important in increasing the yield ofplants from which the roots or leaves are harvested. In horticulture,control of the timing of flowering is important. Horticultural plantswhose flowering may be controlled include lettuce, endive, spinach andvegetable brassicas including cabbage, broccoli and cauliflower, andcarnations and geraniums.

Arabidopsis thaliana is a facultative long day plant, flowering earlyunder long days and late under short days. Because it has a small,well-characterized genome, is relatively easily transformed andregenerated and has a rapid growing cycle, Arabidopsis is an ideal modelplant in which to study flowering and its control.

We have discovered that one of the genes required for this response tophotoperiod is the Late Elongated Hypocotyl or LHY gene. We have foundthat plants carrying dominant gain of function mutations of the LHY geneflower later than their wild-types under long days but earlier thantheir wild-types under short days. We have now cloned and sequenced theLHY gene, which is provided herein, and demonstrated that the mutationcauses the gene to be transcribed at higher levels than the wild-typegene. This suggests that increased expression of LHY delays floweringunder long days.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the present invention there is provided anucleic acid molecule comprising a nucleotide sequence encoding apolypeptide with LHY function. Those skilled in the art will appreciatethat “LHY function” may be used to refer to the ability to influence thetiming of flowering phenotypically like the LHY gene of Arabidopsisthaliana (the timing being substantially unaffected by vernalisation).LHY mutants exhibit delayed flowering under long days, the timing offlowering being substantially unaffected by vernalisation. Also providedis a nucleotide sequence comprising the 5′ non-coding region of a geneencoding a polypeptide with LHY function, preferably includingsubstantially the whole promoter region of the gene, which gene may havethe sequence of a LHY gene of Arabidopsis thaliana.

Further aspects based on the promoter region are disclosed below.However, discussion of mutation and manipulation of nucleic acidaccording to the invention encoding a LHY qene product (e.g. to makemutants etc., transform cells and plants and so on) applies mutatismutandis to promoter nucleic acid according to the present invention.

Nucleic acid according to the present invention may have the sequence ofa LHY gene of Arabidopsis thaliana, including coding and/or non-codingregions, or be a mutant, variant, derivative or allele of the sequenceprovided. Preferred mutants, variants, derivatives and alleles are thosewhich encode a protein which retains a functional characteristic of theprotein encoded by the wild-type gene, especially the ability to repressor delay flowering, for example by means of the regulation of othergenes, as discussed herein.

A mutant, variant, derivative or allele in accordance with the presentinvention may have the ability to affect a physical characteristic of aplant, particularly a flowering characteristic. In various embodiments amutant, variant, derivative or allele represses flowering compared withwild-type on expression in a plant, e.g. compared with the effectobtained using a gene sequence encoding the polypeptide of FIG. 1 (SEQID NO:2). “Repression” of flowering delays, retards, inhibits or slowsit down. In other embodiments, a mutant, variant, derivative or allelepromotes flowering compared with wild-type on expression in a plant,e.g. compared with the effect obtained using a gene sequence encodingthe polypeptide of FIG. 1 (SEQ ID NO:2). “Promotion” of floweringadvances, accelerates or brings it forward in time. Comparison of effecton flowering or other characteristic may be performed in Arabidopsisthaliana, although nucleic acid according to the present invention maybe used in the production of a wide variety of plants and forinfluencing a characteristic thereof.

As discussed further below, over-expression of nucleic acid according tothe present invention may delay flowering while under expression maypromote flowering in a transgenic plant.

Changes to a sequence, to produce a mutant, variant or derivative, maybe by one or more of addition, insertion, deletion or substitution ofone or more nucleotides in the nucleic acid, leading to the addition,insertion, deletion or substitution of one or more amino acids in theencoded polypeptide. Of course, changes to the nucleic acid which makeno difference to the encoded amino acid sequence are included, includingchanges to the non-coding regions such as the promoter or to bindingsites for factors influencing regulation of gene expression.

A preferred nucleic acid sequence for a LHY gene is shown in FIG. 1 (SEQID NO:1), along with the predicted amino acid sequence of a polypeptidewhich has LHY function. Preferred nucleic acid according to the presentinvention encodes the amino acid sequence encoded by the sequence ofnucleotides shown in FIG. 1 (SEQ ID NO:2).

A mutant, allele, variant or derivative amino acid sequence inaccordance with the present invention may include within the sequenceshown in FIG. 1 (SEQ ID NO:2), a single amino acid change with respectto the sequence shown in FIG. 1 (SEQ ID NO:2), or 2, 3, 4, 5, 6, 7, 8,or 9 changes, about 10, 15, 20, 30, 40 or 50 changes, or greater thanabout 50, 60, 70, 80 or 90 changes. In addition to one or more changeswithin the amino acid sequence shown in FIG. 1 (SEQ ID NO:2), a mutant,allele, variant or derivative amino acid sequence may include additionalamino acids at the C-terminus and/or N-terminus.

A sequence related to a sequence specifically disclosed herein shareshomology with that sequence. Homology may be at the nucleotide sequenceand/or amino acid sequence level. Preferably, the nucleic acid and/oramino acid sequence shares homology with the coding sequence (SEQ IDNO:1) or the sequence encoded by the nucleotide sequence of FIG. 1 (SEQID NO:2), preferably at least about 50%, or 60%, or 70%, or 80%homology, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99%homology.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity. Similarity allows for“conservative variation”, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art, or, and this may be preferred, the standard programBestFit, which is part of the Wisconsin Package, Version 8, September1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA,Wisconsin 53711). BestFit makes an optimal alignment of the best segmentof similarity between two sequences. Optimal alignments are found byinserting gaps to maximize the number of matches using the localhomology algorithm of Smith and Waterman

Homology may be over the full-length of the relevant sequence shownherein, or may more preferably be over a contiguous sequence of about orgreater than about 20, 25, 30, 33, 40, 50, 67, 133, 167, 200, 233, 267,300, 333, 400, 450, 500, 550, 600 or more amino acids or codons,compared with the relevant amino acid sequence or nucleotide sequence asthe case may be.

Also provided by an aspect of the present invention is nucleic acidincluding or consisting essentially of a sequence of nucleotidescomplementary to a nucleotide sequence hybridisable with any encodingsequence provided herein. Another way of looking at this would be fornucleic acid according to this aspect to be hybridisable with anucleotide sequence complementary to any encoding sequence providedherein. Of course, DNA is generally double-stranded and blottingtechniques such as Southern hybridisation are often performed followingseparation of the strands without a distinction being drawn betweenwhich of the strands is hybridising. Preferably the hybridisable nucleicacid or its complement encode a product able to influence a physicalcharacteristic of a plant, particularly a flowering characteristic suchas the timing of flowering. Preferred conditions for hybridisation arefamiliar to those skilled in the art, but are generally stringent enoughfor there to be positive hybridisation between the sequences of interestto the exclusion of other sequences.

The nucleic acid, which may contain for example DNA encoding the aminoacid sequence of FIG. 1 (SEQ ID NO:2), as genomic or cDNA, may be in theform of a recombinant and preferably replicable vector, for example aplasmid, cosmid, phage or Agrobacterium binary vector. The nucleic acidmay be under the control of an appropriate promoter or other regulatoryelements for expression in a host cell such as a microbial, e.g.bacterial, or plant cell. In the case of genomic DNA, this may containits own promoter or other regulatory elements and in the case of cDNAthis may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

A vector including nucleic acid according to the present invention neednot include a promoter or other regulatory sequence, particularly if thevector is to be used to introduce the nucleic acid into cells forrecombination into the genome.

Those skilled in the art are well able to construct vectors and designprotocols for recombinant gene expression. Suitable vectors can bechosen or constructed, containing appropriate regulatory sequences,including promoter sequences, terminator fragments, polyadenylationsequences, enhancer sequences, marker genes and other sequences asappropriate. For further details see, for example, Molecular Cloning: aLaboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring HarborLaboratory Press. Many known techniques and protocols for manipulationof nucleic acid, for example in preparation of nucleic acid constructs,mutagenesis, sequencing, introduction of DNA into cells and geneexpression, and analysis of proteins, are described in detail in CurrentProtocols in Molecular Biology, Second Edition, Ausubel et al. eds.,John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubelet al. are incorporated herein by reference. Specific procedures andvectors previously used with wide success upon plants are described byBevan (Nucl. Acids Res. 12, 8711-8721 (1984)) and Guerineau andMullineaux (1993) (Plant transformation and expression vectors. In:Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS ScientificPublishers, pp 121-148).

Selectable genetic markers may be used consisting of chimaeric genesthat confer selectable phenotypes such as resistance to antibiotics suchas kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate,gentamycin, spectinomycin, imidazolinones and glyphosate.

Nucleic acid molecules and vectors according to the present inventionmay be provided isolated and/or purified from their natural environment,in substantially pure or homogeneous form, or free or substantially freeof nucleic acid or genes of the species of interest or origin other thanthe sequence encoding a polypeptide with the required function. Nucleicacid according to the present invention may include CDNA, RNA, genomicDNA and may be wholly or partially synthetic. The term “isolate”encompasses all these possibilities. Where a DNA sequence is specified,e.g. with reference to a figure, unless context requires otherwise theRNA equivalent, with U substituted for T where it occurs, isencompassed.

The present invention also encompasses the expression product of any ofthe nucleic acid sequences disclosed and methods of making theexpression product by expression from encoding nucleic acid thereforeunder suitable conditions, which may be in suitable host cells.Following expression, the product may be isolated from the expressionsystem and may be used as desired, for instance in formulation of acomposition including at least one additional component.

Purified LHY protein, or a fragment, mutant, derivative or variantthereof, e.g. produced recombinantly by expression from encoding nucleicacid therefor, may be used to raise antibodies employing techniqueswhich are standard in the art. Antibodies and polypeptides comprisingantigen-binding fragments of antibodies may be used in identifyinghomologues from other species as discussed further below.

Methods of producing antibodies include immunising a mammal (e.g. human,mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or afragment thereof. Antibodies may be obtained from immunised animalsusing any of a variety of techniques known in the art, and might bescreened, preferably using binding of antibody to antigen of interest.For instance, Western blotting techniques or immunoprecipitation may beused (Armitage et al, 1992, Nature 357: 80-82). Antibodies may bepolyclonal or monoclonal.

As an alternative or supplement to immunising a mammal, antibodies withappropriate binding specificity may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using lambda bacteriophage or filamentous bacteriophage which displayfunctional immunoglobulin binding domains on their surfaces; forinstance see WO92/01047.

Antibodies raised to a polypeptide or peptide can be used in theidentification and/or isolation of homologous polypeptides, and then theencoding genes. Thus, the present invention provides a method ofidentifying or isolating a polypeptide with LHY function (in accordancewith embodiments disclosed herein), comprising screening candidatepolypeptides with a polypeptide comprising the antigen-binding domain ofan antibody (for example whole antibody or a fragment thereof) which isable to bind an LHY polypeptide or fragment, variant or derivativethereof or preferably has binding specificity for such a polypeptide.Specific binding members such as antibodies and polypeptides comprisingantigen binding domains of antibodies that bind and are preferablyspecific for a LHY polypeptide or mutant, variant or derivative thereofrepresent further aspects of the present invention, as do their use andmethods which employ them.

Candidate polypeptides for screening may for instance be the products ofan expression library created using nucleic acid derived from a plant ofinterest, or may be the product of a purification process from a naturalsource.

A polypeptide found to bind the antibody may be isolated and then may besubject to amino acid sequencing. Any suitable technique may be used tosequence the polypeptide either wholly or partially (for instance afragment of the polypeptide may be sequenced). Amino acid sequenceinformation may be used in obtaining nucleic acid encoding thepolypeptide, for instance by designing one or more oligonucleotides(e.g. a degenerate pool of oligonucleotides) for use as probes orprimers in hybridization to candidate nucleic acid, or by searchingcomputer sequence databases, as discussed further below.

A further aspect of the present invention provides a method ofidentifying and cloning LHY homologues from plant species other thanArabidopsis thaliana which method employs a nucleotide sequence derivedfrom that shown in FIG. 1 (SEQ ID NO:1). Sequences derived from thesemay themselves be used in identifying and in cloning other sequences.The nucleotide sequence information provided herein, or any partthereof, particularly in relation to the myb domain, may be used in adata-base search to find homologous sequences, expression products ofwhich can be tested for ability to influence a flowering characteristic.These may have LHY function or the ability to repress flowering(especially under long days), preferably the timing of flowering beingsubstantially unaffected by vernalisation, as disclosed herein.Alternatively, nucleic acid libraries may be screened using techniqueswell known to those skilled in the art and homologous sequences therebyidentified then tested. Thus, a method of obtaining nucleic acid whoseexpression is able to influence a flowering characteristic of a plant isprovided, comprising hybridisation of an oligonucleotide or a nucleicacid molecule comprising such an oligonucleotide to target/candidatenucleic acid. Target or candidate ucleic acid may, for example, comprisea genomic or cDNA library obtainable from an organism known to containor suspected of containing such nucleic acid. Successful hybridisationmay be identified and target/candidate nucleic acid isolated for furtherinvestigation and/or use.

Hybridisation may involve probing nucleic acid and identifying positivehybridisation under suitably stringent conditions (in accordance withknown techniques) and/or use of oligonucleotides as primers in a methodof nucleic acid amplification, such as PCR. For probing, preferredconditions are those which are stringent enough for there to be a simplepattern with a small number of hybridisations identified as positivewhich can be investigated further. It is well known in the art toincrease stringency of hybridisation gradually until only a few positiveclones remain.

As an alternative to probing, though still employing nucleic acidhybridisation, oligonucleotides designed to amplify DNA sequences may beused in PCR reactions or other methods involving amplification ofnucleic acid, using routine procedures. See for instance “PCR protocols;A Guide to Methods and Applications”, Eds. Innis et al, 1990, AcademicPress, New York.

Preferred amino acid sequences suitable for use in the design of probesor PCR primers are sequences conserved (completely, substantially orpartly) between at least two LHY polypeptides able to influence aflowering characteristic, such as timing of flowering. Other preferredprimers are designed to amplify a region including a myb domain.

On the basis of amino acid sequence information oligonucleotide probesor primers may be designed, taking into account the degeneracy of thegenetic code, and, where appropriate, codon usage of the organism fromthe candidate nucleic acid is derived.

Preferably an oligonucleotide in accordance with the present invention,e.g. for use in nucleic acid amplification, has about 10 or fewer codons(e.g. 6, 7 or 8), i.e. is about 30 or fewer nucleotides in length (e.g.18, 21 or 24). Possible primers for amplifying a LHY wild-type or mutantgene include: 5′-ATGGATACTAATACATCT-3′ (SEQ ID NO:3) and5′-CTAGATTTAAAGATATTA-3′ (SEQ ID NO:4). For amplifying the promoter, theprimers LP1(SEQ ID NO:26) and LP2(SEQ ID NO:27) may be used (see below).

Assessment of whether or not a PCR product corresponds to a geneinvolved in the control of flowering may be conducted in various ways. APCR band from such a reaction might contain a complex mix of products.Individual products may be cloned and each one individually screened.They may be analysed by transformation to assess function onintroduction into a plant of interest.

The present invention also extends to nucleic acid encoding a LHYhomologue obtained using a nucleotide sequence derived from that shownin FIG. 1 (SEQ ID NO:1), and uses thereof. No genes showing significanthomology to LHY were identified in public databases, except forExpressed Sequence Tags (ESTs) of unknown function. However, a region ofLHY (between amino acids 18 and 78) (SEQ ID NO:13) showed weak homologyto several DNA binding proteins that contained a MYB domain (Frampton etal, 1989) careful analysis of this portion of the sequence of LHYdemonstrated that this also probably contains a MYB domain. Inparticular, it is predicted to contain three alpha helices as discussedin Example 1 (FIG. 8). Unlike most MYB proteins, LHY contains a singleMYB repeat, although other proteins containing a single MYB repeat werepreviously reported (Baranowski et al. 1994).

The provision of sequence information for the LHY gene of Arabidopsisthaliana enables the obtention of homologous sequences from other plantspecies. In particular, those skilled in the art may isolate LHYanalogues from related, commercially important Brassica species (e.g.Brassica nigra, Brassica napus and Brassica oleraceae), as has been donefor other flowering time genes isolated from Arabidopsis (e.g. CO; WO96/14414).

Thus, included within the scope of the present invention are nucleicacid molecules which encode amino acid sequences which are homologues ofLHY of Arabidopsis thaliana. Homology may be at the nucleic acidsequence or amino acid sequence level. Preferably, the nucleic acid oramino acid sequence shares homology with a sequence of FIG. 1 (SEQ IDNO:1 and SEQ ID NO:2), preferably at least about 50%, or at least about60% or at least about 70% or at least about 80% homology, mostpreferably at least about 90% homology from species other thanArabidopsis thaliana and the encoded polypeptide shares a phenotype withthe Arabidopsis thaliana LHY gene, preferably the ability to influencetiming of flowering. These may promote or delay flowering compared withArabidopsis thaliana LHY and mutants, variants or alleles may promote ordelay flowering compared with wild-type. “Homology” may be used to referto identity.

LHY gene homologues may also be identified from economically importantmonocotyledonous crop plants such as rice and maize. Although genesencoding the same protein in monocotyledonous and dicotyledonous plantsshow relatively little homology at the nucleotide level, amino acidsequences are conserved. In public sequence databases we recentlyidentified several Arabidopsis cDNA clone sequences that were obtainedin random sequencing programmes and share homology with LHY in regionsof the protein that are known to be important for its activity.Similarly, a randomly sequenced rice cDNA showing strong homology to LHYwas identified by homology, and should provide access to the LHY gene ineconomically important cereal plants. By sequencing each of theseclones, studying their expression patterns and examining the effect ofaltering their expression, genes carrying out a similar function to LHYin regulating flowering time are obtainable. Of course, mutants,derivatives and alleles of these sequences are included in the scope ofthe present invention in the same terms as discussed above for the LHYgene.

In certain embodiments, nucleic acid according to the present inventionencodes a polypeptide which has homology with all or part of the aminoacid sequence shown in FIG. 1 (SEQ ID NO:2), in the terms discussedalready above (e.g. for length), which homology is greater over thelength of the relevant part (i.e. fragment) than the homology sharedbetween a respective part of the amino acid sequence of FIG. 1 (SEQ IDNO:2) an EST sequence such as shown in Figure (SEQ ID NO:16 and SEQ IDNO:17), and may be greater than about 5% greater, more preferablygreater than about 10% greater, more preferably greater than about 20%greater, and more preferably greater than about 30% greater. Thus, toexemplify with reference to one embodiment, nucleic acid encoding anamino acid mutant, variant or derivative of the amino acid sequenceshown in FIG. 1 (SEQ ID NO:2) may be provided wherein the encoded aminoacid sequence includes a contiguous sequence of about 100 amino acidswhich has greater homology with a contiguous sequence of 100 amino acidswithin the amino acid sequence of FIG. 1 (SEQ ID NO:2) than anycontiguous sequence of 100 amino acids within an EST sequence such asshown in FIG. 9 (SEQ ID NO:16 and SEQ ID NO:17), preferably greater thanabout 5% greater homology, and so on.

Similarly, nucleic acid according to certain embodiments of the presentinvention may have homology with all or part of the nucleotide sequenceshown in FIG. 1 (SEQ ID NO:1), in the terms discussed already above(e.g. for length), which homology is greater over the length of therelevant part (i.e. fragment) than the homology shared between arespective part of the nucleotide sequence of FIG. 1 (SEQ ID NO:1) andthe encoding nucleotide sequence for an EST sequence such as shown inFIG. 9 (SEQ ID Nos:16 and 17) (accession numbers for which are givenbelow) and may be greater than about 5% greater, more preferably greaterthan about 10% greater, more preferably greater than about 20% greater,and more preferably greater than about 30% greater. Thus, to exemplifywith reference to one embodiment, nucleic acid may be provided inaccordance with the present invention wherein the nucleotide sequenceincludes a contiguous sequence of about 300 nucleotides (or 100 codons)which has greater homology with a contiguous sequence of 300 nucleotideswithin the nucleotide sequence of FIG. 1 (SEQ ID NO:1) than anycontiguous sequence of 100 nucleotides within the coding nucleotidesequence for an EST sequence such as shown in FIG. 9 (SEQ ID NO:16 andSEQ ID NO:17) (accession numbers for which are given below), preferablygreater than about 5% greater homology, and so on.

Nucleic acid according to the present invention may include a nucleicacid sequence encoding a polypeptide which when expressed (e.g. at ahigh level) delays flowering, the timing of flowering beingsubstantially unaffected by vernalisation. The delayed flowering may beunder long days. The delay in flowering caused by LHY is a consequenceof active over-expression of the gene and is therefore distinguishedfrom that previously described for CO and LD in which loss of functionresults in delayed flowering. Expression of the LHY gene product, e.g.from the CaMV35S promoter, causes late flowering, whilst over-expressionof the products of other genes identified in late flowering utants, suchas CO, causes early flowering. The effect of expression fo the LHY genemay arise from the product actively repressing flowering under longdays, or disrupting the functioning of a process required for earlyflowering, such as disruption of the circadian clock.

Vernalisation is low-temperature (e.g. from around −1° C. to around 6°C., usually just above 0° C.) treatment of plant (seedlings) or seed fora period of usually a few weeks or months, probably between about 30days and about 60 days. It is a treatment required by some plant speciesbefore they will break bud or flower, simulating the effect of wintercold.

Also according to the present invention there is provided a plant cellhaving incorporated into its genome a heterologous sequence ofnucleotides as provided by the present invention, under operativecontrol of a regulatory sequence for control of expression. A furtheraspect of the present invention provides a method of making such a plantcell involving introduction of a vector comprising the sequence ofnucleotides into a plant cell and causing or allowing recombinationbetween the vector and the plant cell genome to introduce the sequenceof nucleotides into the genome. A plant may be regenerated from one ormore transformed plant cells.

When introducing a chosen gene construct into a cell, certainconsiderations must be taken into account, well known to those skilledin the art. The nucleic acid to be inserted should be assembled within aconstruct which contains effective regulatory elements which will drivetranscription. There must be available a method of transporting theconstruct into the cell. Once the construct is within the cell membrane,integration into the endogenous chromosomal material either will or willnot occur. Finally, as far as plants are concerned the target cell typemust be such that cells can be regenerated into whole plants.

Plants transformed with the DNA segment containing the sequence may beproduced by standard techniques which are already known for the geneticmanipulation of plants. DNA can be transformed into plant cells usingany suitable technology, such as a disarmed Ti-plasmid vector carried byAgrobacterium exploiting its natural gene transfer ability (EP-A-270355,EP-A-0116718, NAR 12(22) 8711-87215 1984), particle or microprojectilebombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616)microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green etal. (1987) Plant Tissue and Cell Culture, Academic Press),electroporation (EP 290395, WO 8706614 Gelvin Debeyser—see attached)other forms of direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No.4,684,611), liposome mediated DNA uptake (e.g. Freeman et al. Plant CellPhysiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNASU.S.A. 87: 1228 (1990d) Physical methods for the transformation of plantcells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Agrobacterium transformation is widely used by those skilled in the artto transform dicotyledonous species. Recently, there has beensubstantial progress towards the routine production of stable, fertiletransgenic plants in almost all economically relevant monocot plants(Toriyama, et al. (1988) Bio/Technology 6, 1072-1074; Zhang, et al.(1988) Plant Cell Rep. 7, 379-384; Zhang, et al. (1988) Theor Appl Genet76, 835-840; Shimamoto, et al. (1989) Nature 338, 274-276; Datta, et al.(1990) Bio/Technology 8, 736-740; Christou, et al. (1991) Bio/Technology9, 957-962; Peng, et al. (1991) International Rice Research Institute,Manila, Philippines 563-574; Cao, et al. (1992) Plant Cell Rep. 11,585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et al.(1993) Plant Molecular Biology 21, 871-884; Fromm, et al. (1990)Bio/Technology 8, 833-839; Gordon-Kamm, et al. (1990) Plant Cell 2,603-618; D'Halluin, et al. (1992) Plant Cell 4, 1495-1505; Walters, etal. (1992) Plant Molecular Biology 18, 189-200; Koziel, et al. (1993)Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology25, 925-937; Weeks, et al. (1993) Plant Physiology 102, 1077-1084;Somers, et al. (1992) Bio/Technology 10, 1589-1594; WO92/14828). Inparticular, Agrobacterium mediated transformation is now emerging alsoas an highly efficient alternative transformation method in monocots(Hiei et al. (1994) The Plant Journal 6, 271-282).

The generation of fertile transgenic plants has been achieved in thecereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K.(1994) Current Opinion in Biotechnology 5, 158-162.; Vasil, et al.(1992) Bio/Technology 10, 667-674; Vain et al., 1995, BiotechnologyAdvances 13 (4): 653-671; Vasil, 1996, Nature Biotechnology 14 page702).

Microprojectile bombardment, electroporation and direct DNA uptake arepreferred where Agrobacterium is inefficient or ineffective.Alternatively, a combination of different techniques may be employed toenhance the efficiency of the transformation process, eg bombardmentwith Agrobacterium coated microparticles (EP-A-486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP-A-486233).

Following transformation, a plant may be regenerated, e.g. from singlecells, callus tissue or leaf discs, as is standard in the art. Almostany plant can be entirely regenerated from cells, tissues and organs ofthe plant. Available techniques are reviewd in Vasil et al., CellCulture and Somatic Cell Genetics of Plants, Vol I, II and III,Laboratory Procedures and Their Applications, Academic Press, 1984, andWeissbach and Weissbach, Methods for Plant Molecular Biology, AcademicPress, 1989.

The particular choice of a transformation technology will be determinedby its efficiency to transform certain plant species as well as theexperience and preference of the person practising the invention with aparticular methodology of choice. It will be apparent to the skilledperson that the particular choice of a transformation system tointroduce nucleic acid into plant cells is not essential to or alimitation of the invention, nor is the choice of technique for plantregeneration.

DETAILED DESCRIPTION OF THE INVENTION

A LHY gene and a modified version thereof (allele, mutant, variant orderivative thereof), and other nucleic acid provided herein, includingspecies homologues, may be used to affect a physical characteristic,such as a flowering characteristic which may include timing offlowering, in plants. For this purpose nucleic acid such as a vector asdescribed herein may be used for the production of a transgenic plant.Such a plant may possess an altered flowering phenotype, particular interms of timing of flowering, compared with wild-type (that is to say aplant that is wild-type for LHY or the relevant homologue thereof).

The invention further encompasses a host cell transformed with nucleicacid or a vector according to the present invention, especially a plantor a microbial cell. Thus, a host cell, such as a plant cell, includingheterologous nucleic acid according to the present invention isprovided. Within the cell, the nucleic acid may be incorporated withinthe chromosome. There may be more than one heterologous nucleotidesequence per haploid genome.

Also according to the invention there is provided a plant cell havingincorporated into its genome nucleic acid, particularly heterologousnucleic acid, as provided by the present invention, under operativecontrol of a regulatory sequence for control of expression. The codingsequence may be operably linked to one or more regulatory sequenceswhich may be heterologous or foreign to the gene, such as not naturallyassociated with the gene for its expression. The nucleic acid accordingto the invention may be placed under the control of an externallyinducible gene promoter to place expression under the control of theuser.

A suitable inducible promoter is the GST-II-27 gene promoter which hasbeen shown to be induced by certain chemical compounds which can beapplied to growing plants. The promoter is functional in bothmonocotyledons and dicotyledons. It can therefore be used to controlgene expression in a variety of genetically modified plants, includingfield crops such as canola, sunflower, tobacco, sugarbeet, cotton;cereals such as wheat, barley, rice, maize, sorghum; fruit such astomatoes, mangoes, peaches, apples, pears, strawberries, bananas, andmelons; and vegetables such as carrot, lettuce, cabbage and onion. TheGST-II-27 promoter is also suitable for use, in a variety of tissues,including roots, leaves, stems and reproductive tissues.

Other suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV35S) gene promoter that is expressed at a high level in virtually allplant tissues (Benfey et al, 1990a and 1990b); the cauliflower meri 5promoter that is expressed in the vegetative apical meristem as well asseveral well localised positions in the plant body, eg inner phloem,flower primordia, branching points in root and shoot (Medford, 1992;Medford et al, 1991) and the Arabidopsis thaliana LEAFY promoter that isexpressed very early in flower development (Weigel et al, 1992).

Placing nucleic acid according to the present invention, such as a LHYgene or homologue, under the control of an externally inducible genepromoter thus placing the timing of flowering under the control of theuser is advantageous in that, for example, flower production, andsubsequent events such as seed set, may be timed to meet market demands,for example, in cut flowers or decorative flowering pot plants. Delayingflowering in pot plants is advantageous to lengthen the period availablefor transport of the product from the producer to the point of sale andlengthening of the flowering period is an obvious advantage to thepurchaser.

In a further aspect the present invention provides a gene constructcomprising an inducible promoter operatively linked to a nucleotidesequence provided by the present invention, such as the LHY gene ofArabidopsis thaliana, a homologue from another plant species, e.g. aBrassica such as Brassica napus, or any mutant, variant or allelethereof. As discussed, this enables control of expression of the gene.The present invention also provides plants transformed with said geneconstruct and methods comprising introduction of such a construct into aplant cell and/or induction of expression of a construct within a plantcell, by application of a suitable stimulus, an effective exogenousinducer.

The term “inducible” as applied to a promoter is well understood bythose skilled in the art. In essence, expression under the control of aninducible promoter is “switched on” or increased in response to anapplied stimulus. The nature of the stimulus varies between promoters.Some inducible promoters cause little or undetectable levels ofexpression (or no expression) in the absence of the appropriatestimulus. Other inducible promoters cause detectable constitutiveexpression in the absence of the stimulus. Whatever the level ofexpression is in the absence of the stimulus, expression from anyinducible promoter is increased in the presence of the correct stimulus.The preferable situation is where the level of expression increases uponapplication of the relevant stimulus by an amount effective to alter aphenotypic characteristic. Thus an inducible (or “switchable”) promotermay be used which causes a basic level of expression in the absence ofthe stimulus which level is too low to bring about a desired phenotype(and may in fact be zero). Upon application of the stimulus, expressionis increased (or switched on) to a level which brings about the desiredphenotype.

The term “heterologous” may be used to indicate that the gene/sequenceof nucleotides in question have been introduced into said cells of theplant or an ancestor thereof, using genetic engineering, ie by humanintervention. A transgenic plant cell, i.e. transgenic for the nucleicacid in question, may be provided. The transgene may be on anextra-genomic vector or incorporated, preferably stably, into thegenome. A heterologous gene may replace an endogenous equivalent gene,ie one which normally performs the same or a similar function, or theinserted sequence may be additional to the endogenous gene or othersequence. An advantage of introduction of a heterologous gene is theability to place expression of a sequence under the control of apromoter of choice, in order to be able to influence expressionaccording to preference. Furthermore, mutants, variants and derivativesof the wild-type gene, e.g. with higher or lower activity thanwild-type, may be used in place of the endogenous gene. Nucleic acidheterologous, or exogenous or foreign, to a plant cell may benon-naturally occuring in cells of that type, variety or species. Thus,nucleic acid may include a coding sequence of or derived from aparticular type of plant cell or species or variety of plant, placedwithin the context of a plant cell of a different type or species orvariety of plant. A further possibility is for a nucleic acid sequenceto be placed within a cell in which it or a homologue is foundnaturally, but wherein the nucleic acid sequence is linked and/oradjacent to nucleic acid which does not occur naturally within the cell,or cells of that type or species or variety of plant, such as operablylinked to one or more regulatory sequences, such as a promoter sequence,for control of expression. A sequence within a plant or other host cellmay be identifiably heterologous, exogenous or foreign.

Plants which include a plant cell according to the invention are alsoprovided, along with any part or propagule thereof, seed, selfed orhybrid progeny and descendants. A plant according to the presentinvention may be one which does not breed true in one or moreproperties. Plant varieties may be excluded, particularly registrableplant varieties according to Plant Breeders' Rights. It is noted that aplant need not be considered a “plant variety” simply because itcontains stably within its genome a transgene, introduced into a cell ofthe plant or an ancestor thereof.

In addition to a plant, the present invention provides any clone of sucha plant, seed, selfed or hybrid progeny and descendants, and any part ofany of these, such as cuttings, seed. The invention provides any plantpropagule, that is any part which may be used in reproduction orpropagation, sexual or asexual, including cuttings, seed and so on. Alsoencompassed by the invention is a plant which is a sexually or asexuallypropagated off-spring, clone or descendant of such a plant, or any partor propagule of said plant, off-spring, clone or descendant.

The invention further provides a method of influencing or affecting aphysical e.g. flowering characteristic such as the timing of floweringof a plant, including causing or allowing expression of a heterologousnucleic acid sequence as discussed within cells of the plant.

The invention further provides a method of including expression fromnucleic acid encoding the amino acid sequence of FIG. 1 (SEQ ID NO:2),or a mutant, variant, allele or derivative of the sequence, within cellsof a plant (thereby producing the encoded polypeptide), following anearlier step of introduction of the nucleic acid into a cell of theplant or an ancestor thereof. Such a method may influence or affect aflowering characteristic of the plant, such as the timing of flowering.This may be used in combination with any other gene, such as transgenesinvolved in flowering or other phenotypic trait or desirable property.

The principal flowering characteristic which may be altered using thepresent invention is the timing of flowering. (Other physicalcharacteristics of plants may be affected by means of expression fromnucleic acid according to the present invention.) Over-expression of thegene product of the LHY gene leads to delayed flowering, particularlyunder long days (as suggested by the LHY mutant phenotype);under-expression may lead to precocious flowering. This degree ofcontrol is useful to ensure synchronous flowering of male and femaleparent lines in hybrid production, for example. Another use is toadvance or retard the flowering in accordance with the dictates of theclimate so as to extend or reduce the growing season. This may involveuse of anti-sense or sense regulation.

In the present invention, over-expression may be achieved byintroduction of the nucleotide sequence in a sense orientation. Thus,the present invention provides a method of influencing a floweringcharacteristic of a plant, the method comprising causing or allowingexpression of the polypeptide encoded by the nucleotide sequence ofnucleic acid according to the present invention from that nucleic acidwithin cells of the plant.

Under-expression of the gene product polypeptide may be achieved usinganti-sense technology or “sense regulation” (“co-suppression”).

In using anti-sense genes or partial gene sequences to down-regulategene expression, a nucleotide sequence is placed under the control of apromoter in a “reverse orientation” such that transcription yields RNAwhich is complementary to normal mRNA transcribed from the “sense”strand of the target gene. See, for example, Rothstein et al, 1987;Smith et al, (1988) Nature 334, 724-726; Zhang et al, (1992) The PlantCell 4, 1575-1588, English et al., (1996) The Plant Cell 8, 179-188.Antisense technology is also reviewed in Bourque, (1995), Plant Science105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496.

An alternative is to use a copy of all or part of the target geneinserted in sense, that is the same, orientation as the target gene, toachieve reduction in expression of the target gene by co-suppression.See, for example, van der Krol et al., (1990) The Plant Cell 2, 291-299;Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al., (1992)The Plant Cell 4, 1575-1588, and U.S. Pat. No. 5,231,020.

The complete sequence corresponding to the coding sequence (in reverseorientation for anti-sense) need not be used. For example fragments ofsufficient length may be used. It is a routine matter for the personskilled in the art to screen fragments of various sizes and from variousparts of the coding sequence to optimise the level of anti-senseinhibition. It may be advantageous to include the initiating methionineATG codon, and perhaps one or more nucleotides upstream of theinitiating codon. A further possibility is to target a conservedsequence of a gene, e.g. a sequence that is characteristic of one ormore genes, such as a regulatory sequence.

The sequence employed may be about 500 nucleotides or less, possiblyabout 400 nucleotides, about 300 nucleotides, about 200 nucleotides, orabout 100 nucleotides. It may be possible to use oligonucleotides ofmuch shorter lengths, 14-23 nucleotides, although longer fragments, andgenerally even longer than about 500 nucleotides are preferable wherepossible, such as longer than about 600 nucleotides, than about 700nucleotides, than about 800 nucleotides, than about 1000 nucleotides ormore.

It may be preferable that there is complete sequence identity in thesequence used for down-regulation of expression of a target sequence,and the target sequence, though total complementarity or similarity ofsequence is not essential. One or more nucleotides may differ in thesequence used from the target gene. Thus, a sequence employed in adown-regulation of gene expression in accordance with the presentinvention may be a wild-type sequence (e.g. gene) selected from thoseavailable, or a mutant, derivative, variant or allele, by way ofinsertion, addition, deletion or substitution of one or morenucleotides, of such a sequence. The sequence need not include an openreading frame or specify an RNA that would be translatable. It may bepreferred for there to be sufficient homology for the respectiveanti-sense and sense RNA molecules to hybridise. There may be downregulation of gene expression even where there is about 5%, 10%, 15% or20% or more mismatch between the sequence used and the target gene.

Generally, the transcribed nucleic acid may represent a fragment of anLHY gene, such as including a nucleotide sequence shown in FIG. 1 (SEQID NO:1), or the complement thereof, or may be a mutant, derivative,variant or allele thereof, in similar terms as discussed above inrelation to alterations being made to an LHY coding sequence and thehomology of the altered sequence. The homology may be sufficient for thetranscribed anti-sense RNA to hybridise with nucleic acid within cellsof the plant, though irrespective of whether hybridisation takes placethe desired effect is down-regulation of gene expression.

Thus, the present invention also provides a method of influencing aflowering characteristic of a plant, the method including causing orallowing anti-sense transcription from heterologous nucleic acidaccording to the invention within cells of the plant.

The present invention further provides the use of the nucleotidesequence of FIG. 1 (SEQ ID NO:2) or a fragment, mutant, derivative,allele, variant or homologue thereof for down-regulation of geneexpression, particularly down-regulation of expression of an LHY gene orhomologue thereof, preferably in order to influence a physicalcharacteristic of a plant, especially a flowering characteristic such asthe timing of flowering.

Anti-sense regulation may itself be regulated by employing an induciblepromoter in an appropriate construct.

When additional copies of the target gene are inserted in sense, that isthe same, orientation as the target gene, a range of phenotypes isproduced which includes individuals where over-expression occurs andsome where under-expression of protein from the target gene occurs. Whenthe inserted gene is only part of the endogenous gene the number ofunder-expressing individuals in the transgenic population increases. Themechanism by which sense regulation occurs, particularlydown-regulation, is not well-understood. However, this technique iswell-reported in scientific and patent literature and is used routinelyfor gene control. See, for example, van der Krol, 1990; Napoli et al,1990; Zhang et al, 1992.

Again, fragments, mutants and so on may be used in similar terms asdescribed above for use in anti-sense regulation.

Thus, the present invention also provides a method of influencing aflowering characteristic of a plant, the method comprising causing orallowing expression from nucleic acid according to the invention withincells of the plant. This may be used to suppress activity of apolypeptide with ability to influence a flowering characteristic. Herethe activity of the polypeptide is preferably suppressed as a result ofunder-expression within the plant cells.

Late Flowering Caused by Increased Expression of the LHY Gene

As described in Example 1, the lhy mutation causes late flowering underinductive long-day conditions and is caused by increased expression ofthe gene from a CaMV 35S promoter carried by a Ds transposon. This cantherefore be considered as a transcriptional fusion, that was formed invivo via transposition, between the CaMV 35S promoter and the LHY gene.This phenomenon was reported previously for a different gene (Wilson etal, 1996). An in vitro constructed fusion between the CaMV 35S promoterand the LHY gene, such that the LHY gene product is over expressed wouldbe predicted to have the same effect. Example 2 demonstrates thatintroduction of the in vivo fusion between the CaMV 35S promoter and LHYinto wild-type plants delays flowering under long days. This suggeststhat fusions between foreign promoters and the LHY gene could be used todelay flowering.

Causing Early Flowering by Reducing the Activity of LHY

The observation that increased expression of the LHY gene delaysflowering suggests that the normal function of LHY might be to delayflowering, and that increasing the expression of the gene furtherenhances this effect. In this case, inactivating the LHY gene might beexpected to cause early flowering. This could be done by classicalchemical or radiation mutagenesis, by introducing constructs thatexpress antisense copies of the LHY mRNA, by sense strand inactivationsuch as co-suppression or by expressing modified versions of the LHYprotein.

The LHY promoter for which the sequence is given in FIG. 10 (SEQ IDNO:18) may be used in a number of ways, particularly when operablylinked to a heterologous, exogenous or foreign transgene or fragmentthereof. In essence the promoter may be used to regulate expression ofany sequence of interest, its usefulness arising from its regulation ina circadian oscillatory manner.

For instance, a construct including the promoter (and any suitablemutant, variant, allele or derivative thereof, by way of addition,insertion, substitution and/or deletion of one or more nucleotides) maybe used to control expression of a coding sequence or a sequence, eitheranti-sense or sense, for regulation particularly down-regulation ofexpression of a gene.

Thus genes may be expressed or down-regulated in the early morning butnot at times of the day when they, or their down-regulation as the casemay be, are not needed. This allows for energy reserves of a transgenicplant to be conserved. Furthermore, it allows for expression ordown-regulation of genes in a manner which may avoid adverse sideeffects on a transgenic plant which effects may arise if the gene ordown-regulation is constitutive.

An example of this is using a promoter according to the presentinvention to drive transcription of a sequence for down-regulation ofthe Mlo gene (Buschges, et al, 1997, CELL, 88, 695-705). Down-regulationof this barley gene or the equivalent homologue in other speciesprovides broad spectrum mildew disease resistance. However, some allelesof mlo have associated negative effect on plant performance because theyhave a high level of localised necrotic response. Down-regulating mlo inan oscillatory manner may provide the desirable pathogen resistancewithout the undesirable side effect.

Other genes which may advantageously be expressed in a circadianrhythmic fashion using a promoter in accordance with the presentinvention include genes responsible for diverting metabolic resourcesinto sink storage pathways e.g. starch, oil or other storage proteinsynthesis needed to be on or off during the day or night as appropriate,genes for improving night-time photorespiratory activity which are notneeded during the day, gene used to modify photosynthesis during the daybut not needed during the night, genes for protecting a plant fromphotinhibitory conditions, typically high light levels and lowtemperatures that occur often at dawn, and genes for modifyingpigmentation, e.g. chlorophyll, anthocyanins, to achieve a cosmeticallyvaluable change to the pigmentation pattern (e.g. stripey ornamentals).

Thus, according to a further aspect, the present invention provides anucleic acid molecule encoding a LHY gene promoter.

In another aspect, the present invention provides a nucleic acidmolecule encoding a promoter, the promoter including the promotersequence of nucleotides shown in FIG. 10 (SEQ ID NO:18). Instead ofusing the full length promoter sequence, one or more fragments of thesequence shown in FIG. 10 (SEQ ID NO:18) may be used sufficient topromote gene expression in a plant in a circadian rhythymic fashion. Thepromoter may include or consist essentially of a sequence of up to about1740 nucleotides 5′ to coding sequence of the LHY gene in theArabidopsis thaliana chromosome, or an equivalent sequence in anotherspecies. As noted, a fragment of this may be used, such as about 1700nucleotides, 1600 nucleotides, 1500 nucleotides, 1400 nucleotides, 1300nucleotides, 1200 nucleotides, 1100 nucleotides, 1000 nucleotides, 900nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides or 500nucleotides, particularly a fragment within the sequence upstream fromnucleotide 1059 in FIG. 10 (SEQ ID NO:18). The untranslated leadersequence at nucleotides 1059 to 1735 in FIG. 10 (SEQ ID NO:18) may beomitted. Of course, mutants, alleles, variants, derivatives andhomologues may be employed in accordance with the present invention, asdiscussed.

Any of the sequences disclosed in the figures herein for a codingsequence according to the present invention may be used to construct aprobe for use in identification and isolation of a promoter from agenomic library containing a genomic LHY gene (including homologues).Techniques and conditions for such probing are well known in the art andare discussed elsewhere herein. To find minimal elements or motifsresponsible for temporal regulation, restriction enzyme or nucleases maybe used to digest a nucleic acid molecule, followed by an appropriateassay (for example using a reporter gene such as luciferase) todetermine the sequence required. A preferred embodiment of the presentinvention provides a nucleic acid isolate with the minimal nucleotidesequence shown in FIG. 10 (SEQ ID NO:18) required for circadian rhythmicpromoter activity.

As noted, the promoter may include one or more sequence motifs orelements conferring circadian rhythmic regulatory control of expression.Other regulatory sequences may be included, for instance as identifiedby mutation or digest assay in an appropriate expression system or bysequence comparison with available information, e.g. using a computer tosearch on-line databases.

By “promoter” is meant a sequence of nucleotides from whichtranscription may be initiated of DNA operably linked downstream (i.e.in the 3′ direction on the sense strand of double-stranded DNA).

“Operably linked” means joined as part of the same nucleic acidmolecule, suitably positioned and oriented for transcription to beinitiated from the promoter. DNA operably linked to a promoter is “undertranscriptional initiation regulation” of the promoter.

The present invention extends to a promoter which has a nucleotidesequence which is allele, mutant, variant or derivative, by way ofnucleotide addition, insertion, substitution or deletion of a promotersequence as provided herein. Preferred levels of sequence homology witha provided sequence may be analogous to those set out above for encodingnucleic acid and polypeptides according to the present invention.Systematic or random mutagenesis of nucleic acid to make an alterationto the nucleotide sequence may be performed using any technique known tothose skilled in the art. One or more alterations to a promoter sequenceaccording to the present invention may increase or decrease promoteractivity, or increase or decrease the magnitude of the effect of asubstance able to modulate the promoter activity.

“Promoter activity” is used to refer to ability to initiatetranscription. The level of promoter activity is quantifiable forinstance by assessment of the amount of mRNA produced by transcriptionfrom the promoter or by assessment of the amount of protein productproduced by translation of mRNA produced by transcription from thepromoter. The amount of a specific mRNA present in an expression systemmay be determined for example using specific oligonucleotides which areable to hybridise with the mRNA and which are labelled or may be used ina specific amplification reaction such as the polymerase chain reaction.Use of a reporter gene facilitates determination of promoter activity byreference to protein production.

Further provided by the present invention is a nucleic acid constructincluding a promoter region as provided or a fragment, mutant, allele,derivative or variant thereof able to promoter transcription, operablylinked to a heterologous gene, e.g. a coding sequence. A “heterologous”or “exogenous” gene is generally not a modified form of LHY. The genemay be transcribed into mRNA which may be translated into a peptide orpolypeptide product which may be detected and preferably quantitatedfollowing expression. A gene whose encoded product may be assayedfollowing expression is termed a “reporter gene”, i.e. a gene which“reports” on promoter activity.

The reporter gene preferably encodes an enzyme which catalyses areaction which produces a detectable signal, preferably a visuallydetectable signal, such as a coloured product. The presence and/oramount of gene product resulting from expression from the reporter genemay be determined using a molecule able to bind the product, such as anantibody or fragment thereof. The binding molecule may be labelleddirectly or indirectly using any standard technique.

Those skilled in the art are well aware of a multitude of possiblereporter genes and assay techniques which may be used to determine geneactivity. Examples of reporter genes commonly used in plants include thefirefly luciferase gene (Millar, et al, 1992, The Plant Cell 4,1075-1087) and the E.coli uidA gene (Jefferson, et al, 1987, EMBO J.6,3901-3907). Any suitable reporter/assay may be used and it should beappreciated that no particular choice is essential to or a limitation ofthe present invention.

A promoter construct may be introduced into a cell using any techniquepreviously described, e.g. to produce a stable cell line containing theconstruct integrated into the genome or for transient expression. Cellsand plants etc. containing a promoter or construct and other methods anduses involving such as promoter or construct in accordance with thepresent invention are provided as aspects of the invention in the sameterms as discussed above for other nucleic acid according to theinvention.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

In the FIGURES:

FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) according to oneembodiment of the invention, being the sequence of the LHY ORF, obtainedfrom Arabidopsis thaliana, with the predicted amino acid sequence (SEQID NO:2) shown below the coding region of said nucleotide sequence. Theregion underlined shows sequence which has been deleted in lhy mutants.The position of the insertion is indicated with a triangle.

FIG. 2: A graph showing the hypocotyl lengths of lhy mutant seedlings(2-4 leaves) and wild type grown on soil under different day lengths.Hypocotyl length in mm is shown for seedlings grown in the dark andunder days of length 10, 16 and 24 hours light. Shaded bars are for lhymutant seedlings; unshaded are for wild type.

FIG. 3(A-B): The sequence of IPCR fragments flanking the Ds in the lhymutant. FIG. 3A: IPCR fragment derived from the 3′ end of Ds (SEQ IDNO:5). FIG. 3B: IPCR fragment derived from the 5′ end of Ds (SEQ IDNO:6).

FIG. 4: A restriction enzyme map of the wild type LHY genomic locus andthe deletion present in the lhy mutant.

FIG. 5(A-B): The T-DNA constructs that were used in the complementationexperiments and contain genomic DNA from the region surrounding the LHYgene.

FIG. 6A: Homology of the first 80 amino acids of LHY (SEQ ID NO:7) tothe first alpha helix in the TEA domain of the peptides TEC1 (SEQ IDNO:8) and TEF1 (SEQ ID NO:9).

FIG. 6B: The homology of LHY to the myb domains in YCS3 (SEQ ID NO:10)and BAS1 (SEQ ID NO:11 and SEQ ID NO:12).

FIG. 7: The consensus sequence for a myb domain described in Frampton etal. (1989) with the corresponding LHY sequence underneath. Hash symbolsrepresent hydrophobic residues and dollar symbols represent chargedamino acids.

FIG. 8: The predicted alpha helices of the potential myb domain in LHY(SEQ ID NO:13). H indicates that the residue is likely to to be part ofan alpha helix, and L indicates that the residue is likely to be part ofa loop. The probability of forming a helix is shown below (Prob. H: 0=avery low probability; 9+a very high probability). The structure waspredicted using the software Predict Protein (estimated to be greaterthan 70% accurate) (Rost et al. (1996))

FIG. 9: Two ESTs (SEQ ID NO:16 and SEQ ID NO:17) identified in thedatabase which have homology to the LHY gene (SEQ ID NO:14 and SEQ IDNO:15).

FIG. 10 shows the genomic sequence of the promoter and untranslatedleader of the LHY gene (SEQ ID NO:18). The ATG start codon (bases1736-1738) of the LHY open reading frame is underlined. The longest LHYcDNA obtained starts at position 1059 (in bold and double underlined),indicating that the first 1059 bases include promoter sequence whilethat from 1059 to the ATG start codon encode the untranslated leader.There are sequences present in the genomic sequence of the untranslatedleader and absent from the cDNA, which are introns that are spliced fromthe mRNA. The likely sequences of these introns are marked with italicsin FIG. 10 (1228-1398; 1434-1592). The sequence GGATCC (16-21) denotesthe BamHI site used to clone the promoter.

FIG. 11 shows the nucleotide sequence of EST 162I3T7, Accession no.R30439 (SEQ ID NO:19).

EXAMPLE 1

Cloning and Analysis of a LHY Gene Lambda and CDNA Libraries

A Lambda gt library containing Arabidopsis genomic DNA was obtained fromDr. N. Harberd (Whitelam et al. 1993). A cosmid library containingArabidopsis thaliana genomic fragments in a plant transformable vector(T-DNA vector 04541) was obtained from Dr. C. Lister. 3 cDNA librarieswere obtained from the Arabidopsis stock centre in Koln.

Identification of lhy in a Transposon Mutagenesis Screen.

The lhy mutant was identified in a two component Ac/Ds transposonmutagenesis screen, as described by Long et al. (1993). It incorporateda modified stable Ac element containing a CaMV 35S promotor driventransposase gene. This stable Ac was used to mobilise a modified Dselement. The modified Ds contained a hygromycin resistance gene to alloweasy identification of plants containing the Ds, and a CaMV 35S promotorreading out of the transposon at the 5′ end. It has been shown thatinsertion of the Ds element into the proximity of a native gene cancause overexpression of that gene by the production of a gene fusionbetween the CaMV 35S promoter and the native gene (Wilson et al. 1996).

The mutagenesis strategy is can be summarised as follows: Plantshomozygous for the Ds (marked by hygromycin resistance) were crossedwith plants homozygous for the stable Ac containing transposase source(marked by β-glucoronidase gene. The F1 heterozygous plants were thenallowed to self fertilise. In this generation the Ds may excise from thestreptomycin gene in the T-DNA due to the presence of the transposasesource in the Ac. occasionally it will reinsert somewhere in the genomeand disrupt a native gene. F2 plants were selected for seedlings whichcontained an excised and reinserted, i.e. transposed, Ds, by selectinghygromycin and Streptomyin resistant seedlings. Such plants were selfedand the F3 plants were screened for mutations. This was a segregatingpopulation of plants ccontaining Ds and Ac. If the Ds has inserted intoa gene then the progeny will also segregate with a mutant phenotype.

lhy mutants were identified in the F3 generation as Late floweringplants.

Description of the lhy Mutant Phenotype

When lhy mutants are grown under long days they flower later than wildtype. As well as flowering late lhy mutants show other pleiotropiceffects; their growth rate is reduced compared to wild type, lhyseedlings have an overall etiolated appearance, and if grown in darknessfor three days have an exaggerated apical hook (similar to those ofethylene over expression mutants eg. etol van der Straeten et al.1993).

When grown under long day conditions lhy mutants flower with morevegetative leaves and later than wild type, as shown in Table 1. This isa completely dominant effect, with heterozygous plants flowering withexactly the same leaf number and at the same time as the homozygousplants. Under short day conditions lhy flowers slightly earlier thanwild type making its response like that of other day neutral floweringtime mutants eg. co (Koorneef et al.1991). However lhy produces leavesat a slower rate than wild type under both long and short days, so interms of days to flower the lhy mutant flowers considerably later thanwild-type under long days and at a similar time to wild-type under shortdays (Table 1). When the lhy mutant was grown under continuous light itflowered later than wild type.

lhy and wild type were vernalised for eight weeks at 4° C. and thenmoved to long day conditions to determine whether the flowering time oflhy was reverted to that of wild type. lhy flowered with an average of9.96 leaves showing that although flowering occurs earlier than nonvernalised plants, the effect of the mutation is not corrected byvernalisation (wild-type plants flower with an average of 6.52 leaves).

The rate of leaf development of lhy mutants and wild type was measuredunder long and short day lengths. Leaf counts were conducted every threedays and the number of leaves over 3 mm in length were recorded. Therate of leaf development of wild type plants was constant under long andshort days but that of the lhy mutant was much slower, than wild typeunder both conditions (Table 2). However when the plants were grownunder continuous light the rate of leaf development in the lhy mutantincreased to that of wild type.

The hypocotyl length of lhy and wt plants grown on soil, in differentlight conditions, was measured when the plants were at the two to fourleaf stage. These were compared to plants grown in the dark on minimalMS media (FIG. 2). In wild type the hypocotyl length decreased noticablywhen plants were exposed to light. With lhy the hypocotyl does notelongate as much as wild type in dark conditions, but when lhy is grownunder short days it has an elongated hypocotyl compared to wild type.This elongation seems to be dependant on the amount of light theseedlings receives because under continuous light the hypocotyl lengthsof the mutant was similar to that of wild type.

The lhy mutant shows defective regulation of processes that areregulated by the endogenous circadian clock. In Arabidopsis rhythmicleaf movements and rhythmic expression of the CAB2 or CCR2 genes areoften used to monitor the function of the circadian clock. We have shownthat in lhy mutants leaf movements and the expression of the CAB2 andCCR2 genes are arrythmic, suggesting that in the lhy mutant thefunctioning of the circadian clock has been disrupted. The lateflowering of the lhy mutant under long days may also be a consequence ofdisruption of clock function.

Cloning of the lhy Gene

To isolate the lhy gene, fragments of DNA adjacent to the Ds wereobtained using Inverse PCR (IPCR). Primers specific for sequence at thetermini of the Ds were used to amplify fragments by PCR. Pieces of DNAcontaining Ds sequence and small regions of the flanking genomicsequence were isolated. A second round of PCR was then performed with asecond set of primers that were predicted to anneal to the PCR productto check that the correct fragment had been amplified. A fragmentestimated to be 700 bp long was isolated 5′ to the Ds, using BstY1 todigest mutant genomic DNA and the primers D74 and B34 for the initailamplification. Primers E4 and D73 were used for the second round. Afragment 200 bp long was isolated 3′ to the Ds, by digesting genomic DNAwith Sau3A and using the primers B39 and B38 for the initial PCR andthen the primers D71 and DL4 for the second round. The IPCR fragmentswere than sequenced as seen in FIG. 3 (SEQ ID NO:5 and SEQ ID NO:6), andit was shown to contain the predicted Ds sequence.

Isolation of Larger Fragments of DNA in the Vicinity of the Ds.

A lambda phage gt library made from wild-type genomic DNA was probedusing the IPCR fragment 5′ to the Ds (SEQ ID NO:6). A lambda clonecontaining an llkb insert which strongly hybridised to the fragment 5′to the Ds was isolated. However when the IPCR fragment 3′ to the Ds (SEQID NO:5) was used as a probe no hybridisation occurred. From this, andsouthern analysis of mutant and wild type DNA, it was concluded that a7-8 kb deletion had occurred adjacent to the Ds. A genomic clonespanning this deletion was isolated from a cosmid library of wild-typeDNA, and the region spanning the deletion was mapped with restrictionenzymes (FIG. 4).

Identifying Genes in the Region Surrounding the Ds.

Three cDNAs were identified as hybridising to the region of the Ds andthe deletion. In the mutant, the DNA encoding one of them had beentotally deleted, the 3′ end of a second was deleted and a small regionof the 5′ end of the third was deleted. It was concluded that the thirdcDNA was the most promising candidate for the LHY gene because only asmall region of it had been deleted in the mutant and the Ds element waslocated at the 5′ end of the gene in the orientation such that the CaMV35S promoter within the transposon could transcribe the gene.

To identify which genes were causing the phenotype a complementationexperiment was undertaken by introducing 3 constructs into wild typeplants and one into the lhy mutant, to determine which of the candidategenes was the cause of the lhy mutation. Construct A contained DNA fromwild-type plants that spanned the deletion and was obtained from acosmid library. The other three constructs were obtained from DNA fromthe lhy mutant. Construct B contained the Ds and a region 5′ to theinsertion, Construct C contained the Ds and a region 3′ to the insertionand construct D spanned the Ds and regions on both sides of the element(see FIG. 5). It was found that when constructs B and D were introducedinto wild type plants a lhy mutant phenotype was recreated. See Example2.

Gene Structure

The transformation results showed that a genomic region approximately 4kb 5′ to the Ds together with the adjacent Ds element is sufficient tocause the lhy mutant phenotype. This indicated that the cDNA encoded bythis region, which hybridised to fragments 5′ to the Ds, encoded the lhygene. Also, as the mutation is dominant, it suggested that the lhy genemight be overexpressed in the mutant from the CaMV 35S promoter locatedat the 5′ end of the Ds. The candidate cDNA was sequenced and apredicted 645 amino acid protein was identified (FIG. 1 (SEQ ID NO:1 andSEQ ID NO:2)).

When the predicted LHY protein was compared to known protein sequencesin the Swissprot database by a fasta homology search, the first 80 aminoacids showed homology to the first alpha helix of the TEA domain of TEC1(SEQ ID NO:8) and TEF1 (SEQ ID NO:9) around the region of the firstalpha helix and weak homology to a number of myb DNA binding proteins(FIG. 6). The yeast myb proteins YCS3 (SEQ ID NO:10) (Accession no.P25357), showed 14/41 identity and 24/41 similarity to the myb domain inLHY and the first myb domain in BAS1 (SEQ ID NO:11 and SEQ ID NO:12),(Accesion no. P22035) showed 12/38 identity and 21/38 similarity to themyb domain in LHY.

A classical myb domain consists of three myb repeats, each myb repeatcontains three alpha helices which are defined by evenly spacedtryptophan residues. The first helix is thought to be involved in aprotein-protein interaction, with the second and third showing ahelix-turn-helix DNA binding motif. Not all myb proteins have thisclassical structure, some only have one or two myb repeats. Also thethird tryptophan in the third alpha helix is not always conservered. Atthe amino acid level myb domains are not strongly conserved, it is theoverall nature of the sequence which is important (Frampton et al.1989), they should have correctly spaced hydrophobic amino acids andthree alpha helicies.

The predicted LHY protein only contains a single myb repeat (amino acidresidues 18-78) (SEQ ID NO:13) which has the first two tryptophanresidues with the correct spacing as can be seen in FIG. 7 (fromFrampton et al 1989) but the third tryptophan is absent. The amino acidsequence in this predicted myb domain was analysed with a structureprediction package (predict protein) available on the Internet at theEMBL-Heidelberg web site. For these 60 amino acids there was the highestprediction of three alpha helicies (FIG. 8), supporting the presence ofa single myb domain in LHY. The presence of a myb domain suggests thatLHY functions by binding to DNA, and might therefore regulate theexpression of other genes.

Comparison of lhy to Other Sequences in the Databases.

The predicted protein encoded by the LHY gene was compared to othersequences in the Gen-bank Expressed Sequence Tags (EST) database usingthe Tfasta software available on the Wisconsin software package. Thisprogram translates all the sequences in the databases into possibleproteins (using all six open reading frames) and comparing them to thesequence in question. One EST showed 100% homology to the 3′ end of theLHY transcript (EST clone 162I3T7, Accession no. R30439). This isprobably a truncated cDNA of the LHY gene. Its sequence is shown in FIG.11 (SEQ ID NO:19). It is only 447 bp. A second (SEQ ID NO:16) showedsignificant homology (54% identity, 93% similarity) to the predicted mybDNA binding region in lhy. (EST 157C23T7, Accession no. T88489) Thecomplete sequence of this second EST has not been elucidated yet so itis unknown whether it has homology to LHY in other parts of the gene(see FIG. 9A).

LHY also showed homology (34% identity, 93% homology) to a rice EST (SEQID NO:17). EST OSS15442A (Accesion no. D48887) showed two regions ofstrong homology to the 3′ end of the gene, indicating it might be atruncated cDNA. (FIG. 9B)

Expression of the lhy Gene

RNA from lhy and wild type plants was blotted, onto nitrocellulose andprobed with the LHY gene. The levels of transcript in lhy was at a muchhigher level than wild type. To test whether this expression was due toa gene fusion between the gene and the CaMV 35S promoter, RT-PCR wasconducted on a sample of RNA from lhy using primers specific for the 5′end of the Ds and the LHY transcript. A fragment of DNA was producedwhich corresponded to the cDNA sequence. This was confirmed by Southernanalysis using the LHY gene as a probe.

Native LHY Expression.

The lhy mutant showed a daylength insensitive phenotype, and thereforethere was a possibility that LHY expression is controlled by daylength.RNA was extracted from plants which had been grown under long days (16hours light) and short days (10 hours light) and harvested every twohours from the beginning of the light period for twenty four hours andthen the plants were put into total darkness for a further 48 hours.This analysis demonstrated that the LHY transcript is likely to becircadian rhythm controlled, increasing in the night to a peak when thelights come on and then decreasing slowly. This cycling continues in theabsence of light indicating that the gene is circadian rhythmcontrolled.

The transcript levels were compared for long and short day grown plantsand were found to be higher and lasted for longer under short dayconditions than under long days. However the transcript levels in wildtype never reached the levels of that in lhy. It remains to be testedwhether the late flowering in lhy is due to higher expression of LHY orhaving the transcript present for a longer period.

To determine when LHY is first expressed developmentally, seeds weresown in MS minimal media under LD conditions and harvested at 2 hoursafter the light came on, once a day for eight days. It was found thatwith the germinating seeds the transcript was detectable after two days,indicating that it is first expressed early in seedling development.Three batches of seeds were also germinated in total darkness for twoweeks and the first lot harvested immediately, the second lot after 2hours light, and the third lot after 24 hours of a Long day cycle. Notranscript was detected in the first two samples and faint expressionwas detected in the third sample, indicating the presence of light isneeded to switch the transcript on.

Location of LHY in the Genome

The location of LHY was demonstrated by showing that the CAPS markers(Konieczny and Ausubel, 1993) PVV4 and NCC1 are genetically linked tothe mutation. LHY was mapped 1.8 cM from the marker PVV4. No mutationaffecting flowering time was previously mapped to this position.

METHODS

Growth Conditions and Measurement of Flowering Time

Flowering time was measured under defined conditions by growing plantsin Sanyo Gallenkamp Controlled Environment rooms at 20° C. Short dayscomprised a photoperiod of 10 hours lit with 400 Watt metal halide powerstar lamps supplemented with 100 watt tungsten halide lamps. Thisprovided a level of photosynthetically active radiation (PAR) of 113.7μmoles photons m⁻²s⁻¹ and a red:far red light ration of 2.41. A similarcabinet and lamps were used for the long day. The photoperiod was for 10hours under the same conditions used for short days and extended for afurther 8 hours using only the tungsten halide lamps. In this cabinetthe combination of lamps used for the 10 hour period provided a PAR of92.9 μmoles photons m⁻²s⁻¹ and a red:far red ratio of 1.49. The 8 hourextension produced PAR of 14.27 μmoles m⁻²s⁻¹ and a red:far-red ratio of0.66.

The flowering times of large populations of plants were measured in thegreenhouse. In the summer the plants were simply grown in sunlight. Inwinter supplementary light was provided so that the minumum daylengthwas 16 hours.

To measure flowering time, seeds were placed at 4° C. on wet filterpaper for 4 days to break dormancy and were then sown on soil.Germinating seedlings were usually covered with cling film or propagatorlids for the first 1-2 weeks to prevent dehydration. Flowering time wasmeasured by counting the number of leaves, excluding the cotyledons, inthe rosette at the time the flower bud was visible. Leaf numbers areshown with the standard error at 95% confidence limits. The number ofdays from sowing to the appearance of the flower bud was also recorded,but is not shown.

Plant Material

The standard wild-type genotype used was Arabidopsis thaliana Landsbergerecta.

Isolation of Plant Genomic DNA.

Plant genomic DNA was isolated from glasshouse grown plants essentiallyas described by Tai and Tanksley, Plant Mol. Biol. Rep. 8: 297-303(1991), except that the tissue was ground in liquid nitrogen and theRNase step omitted. Large-scale (2.5-5 g leaves) and miniprep (3-4leaves) DNA was prepared using this method.

Gel Blotting and Hybridisation Conditions.

Gel transfer to Hybond-N, hybridisation and washing conditions wereaccording to the manufacturer's instructions, except that DNA was fixedto the filters by UV Stratalinker treatment (1200 uJ×100; Stratagene)and/or baked at 80° C. for 2 h. Radiolabelled DNA was prepared by randomhexamer labelling.

Inverse Polymerase Chain Reaction (IPCR)

IPCR was used to isolate DNA adjacent to the Ds. The DNA from the lhymutant was cleaved using the restriction enzymes Bst Y1 for the 5′ endand Sau 3A for the 3′ end and then treated as described in Long et al.(1993) The Primers used to amplify the DNA at the 5′ end of the Ds werethe same as those described in Wilson et al. 1996 (D74 and B34 for thefirst round and E4 and D73 in the second round) For the 3′ end primersB38 (5′-GATATACCGGTAACGAAAACGAACGG) (SEQ ID NO:20) and B39(5′-TTCGTTTCCGTCCCGCAAGTTAAATA) (SEQ ID NO:21) were used in the firstround and primers D71 (5′-CGTTACCGACCGTTTTTCATCCCTA) (SEQ ID NO:22) andD75 (5′-ACGAACGGGATAAATACGGTAATC) (SEQ ID NO:23) were used in the secondround.

RNA Extractions

RNA was extracted using a method which is a modified version of thatdescribed by Stiekma et al (1988). Approximately 5 g of tissue frozen inliquid nitrogen was ground in a coffee grinder and extracted with amixture of 15 ml of phenol and 15 ml of extraction buffer (50 mM TrispH8, 1 mM EDTA, 1% SDS). The mixture was shaken, centrifuged and 25 mlof the aqueous layer recovered. This was then shaken vigorously with amixture of 0.7 ml 4M sodium chloride, 10 ml phenol and 10 ml ofchloroform. The aqueous layer was recovered after centrifugation andextracted with 25 ml of chloroform. The RNA was then precipitated from25 ml of the aqueous layer by the addition of 2 ml of 10 M LiCL, and theprecipitate recovered by centrifugation. The pellet was dissolved in 2ml DEPC water and the RNA precipitated by the addition of 0.2 ml of 4Msodium chloride and 4 ml of ethanol. After centrifugation the pellet wasdissolved in 0.5 ml of DEPC water and the RNA concentration determined.

DNA Extractions

Arabidopsis DNA was performed by a CTAB extraction method described byDean et al (1992).

Isolation of cDNA by RT-PCR

Total RNA was isolated from whole seedlings at the 2-3 leaf stagegrowing under long days in the greenhouse. For first strand cDNAsynthesis, 10 ug of RNA in a volume of 10 ul was heated to 65° C. for 3minutes, and then quickly cooled on ice. 10 ul of reaction mix was madecontaining 1 ul of RNAsin, 1 ul of standard dT₁₇-adapter primer (1ug/ul; Frohman et al, 1988), 4 ul of S×reverse transcriptase buffer (250mM TrisHCl pH8.3, 375 mM KCl, 15 mM MgCl₂), 2 ul DTT (100 mM), 1 ul dNTP(20 mM), 1 ul reverse transcriptase (200 units, M-MLV Gibco). Thisreaction mix was then added to the RNA creating a final volume of 20 ul.The mixture was incubated at 42° C. for 2 hours and then diluted to 200ul with water.

10 ul of the diluted first strand synthesis reaction was added to 90 ulof PCR mix containing 4 ul 2.5 mM dNTP, 10 ul 10×PCR buffer (Boehringerplus Mg), 1 ul of a 100 ng/ul solution of each of the primers, 73.7 ulof water and 0.3 ul of 5 units/ul Taq polymerase (Boehringer or CetusAmplitaq). The primers used D73 (5′-GTTAGTTTTATCCCGATCGATTTCGA) (SEQ IDNO:24) and CaRIC7F (5′-ACCGCTTTGATTGAGAAGCTG) (SEQ ID NO:25). Thereaction was performed at 94° C. for 1 minute, 34 cycles of 55° C. for 1minute, 72° C. for 2 minutes and then finally at 72° C. for 10 minutes.

20 ul of the reaction was separated through an agarose gel, and thepresence of a fragment of the expected size was demonstrated afterstaining with ethidium bromide. The DNA was transferred to a filter, andthe fragment of interest was shown to hybridise to a short DNA fragmentderived from the LHY gene.

DNA Sequencing

The Sanger method was used to sequence fragments of interest inserted ina Bluescript plasmid vector. Reactions were performed using a Sequenasekit (United States Biochemical Corporation).

Screening Phage and Cosmid Libraries

A lysate of the cosmid library (Olszewski and Ausubel, 1988) was used toinfect E. coli DH5 alpha, and twenty thousand colonies were screenedwith the probes described in the text. Three cDNA libraries werescreened to try to identify a CO cDNA. The number of plaques screenedwere 5×10⁵ from the “aerial parts” library (supplied by EC ArabidopsisStock Center, MPI, Cologne), 3×10⁵ plaques of a library made from plantsgrowing in sterile beakers (supplied by the EC Arabidopsis Stock Center)and 1×10⁶ plaques of the CD4-71-PRL2 library (supplied by theArabidopsis Biological Resource Center at Ohio State University).

Transformation of Arabidopsis

The cosmids containing DNA from the vicinity of LHY were mobilised intoAgrobacterium tumefaciens C58C1, and the T-DNA introduced intoArabidopsis plants as described by Valvekens et al, 1988. Roots ofplants grown in vitro were isolated and grown on callus-inducing medium(Valvekens et al, 1988) for 2 days. The roots were then cut into shortsegments and co-cultivated with Agrobacterium tumefaciens carrying theplasmid of interest. The root explants were dried on blotting paper andplaced onto callus-inducing medium for 2-3 days. The Agrobacterium werewashed off, the roots dried and placed onto shoot inducing medium(Valvekens et al, 1988) containing vancomycin to kill the Agrobacteriumand kanamycin to select for transformed plant cells. After approximately6 weeks green calli on the roots start to produce shoots. These areremoved and placed in petri dishes or magenta pots containinggermination medium (Valvekens.et al, 1988). These plants produce seedsin the magenta pots. These are then sown on germination mediumcontaining kanamycin to identify transformed seedlings containing thetransgene (Valvekens et al, 1988).

EXAMPLE 2

Delaying Flowering of Wild-type Plants by Introducing a Copy of the LHYGene Fused to the CaMV 35S Promoter

As described in Example 1, introducing constructs B or D (FIG. 5) intowild-type plants was sufficient to cause the lhy mutant phenotype. Thiswas used to identify the LHY gene, because it indicated that the genemust be located on the region of overlap between the two cosmids.Sequence and transcript analysis demonstrated that LHY was the only genelocated in this interval, and that it was over-expressed in the mutant.Introduction of a fusion of the CaMV 35S promoter to the LHY gene intowild-type plants is therefore sufficient to delay flowering, and thisfusion causes the mutant phenotype even in the presence of two copies ofthe wild-type gene.

The lhy mutation has other effects on plant growth and development, aswell as delaying flowering time. For example, it causes a reduction inchlorophyll content and an elongated hypocotyl. The use of promotersother than the CaMV35S promoter to drive over-expression of LHY, orregulation of the timing of over-expression of LHY mAY allow theseparation of the repression of flowering from the other pleiotropiceffects. For example the meri5 (Medford et al, 1991) orshootmeristemless (Long et al, 1996) promoters could be used to driveLHY expression specifically in the shoot meristem. Assuming that LHYencodes a transcription factor, a protein fusion between the LHY geneand a steroid binding site, such as that of the rat glucorticoidreceptor (Lloyd et al, 1994), and expression of the fusion from the CaMV35S promoter would LHY activity to be regulated. This would allowover-expression of the gene to be activated at certain times indevelopment, and therefore repression of flowering to be initiated laterin development. This might, for example, enable flowering to be delayedwithout the effects of the mutation on hypocotyl elongation.

The genomic sequence of the promoter and untranslated leader of the LHYgene is shown in FIG. 10 (SEQ ID NO:18).

The ATG start codon (bases 1740-1742) of the LHY open reading frame isunderlined. The longest LHY CDNA that has been obtained starts atposition 1079 (in bold and underlined); indicating that the the first1079 bases probably encode promoter sequence while that from 1079 to theATG start codon encode the untranslated leader. The region shown initalics from position 1225 to 1370 is present in the genomic sequencebut not the cDNA and represents an intron within the untranslated leadersequence.

The promoter and untranslated leader were cloned by using PCR to amplifythe region from a plasmid carrying a larger fragment of the LHY gene. Aproof reading polymerase was used to reduce the error rate duringamplification. The primers used were LP1 (CATCAACGTAGGGATCCGTGAAATAT)(SEQ ID NO:26) which anneals at the 5′ end of the promoter region frombases 5 to 30 in the sequence shown in FIG. 10. The bases underlined(16-21) represent the BamHI site that was subsequently used to clone theamplified fragment, and those bases double underlined were errorsintroduced into the LP1 primer to introduce the BamHI site. The secondprimer used was LP2 (GGACTAGTAACAGGACCGGTTGCAGCTATTC) (SEQ ID NO:27)which anneals immediately upstream of the ATG and introduces a SpeIsite. A fragment of the expected size was amplified with these primersand inserted into Bluescript vector cleaved with SpeI and BamHI. The DNAsequence of the cloned fragment was then determined and found to be thatexpected.

The promoter may be used to drive gene expression in acircadian-regulated manner similar to that shown by the LHY gene. Forinstance, it may be used to drive expression of firefly luciferase. Thevector VIP1lomeFFLuc which was previously described by Anderson et al(1994) and Anderson and Kay (1995) may be used for this. The vectorcontains BamHI and HindIII sites upstream of the luciferase gene.Bluescript plasmid carrying the LHY promoter fragment described above iscleaved with SpeI and partially filled in with Klenow polymerase to makeit compatible with a partially filled in HindIII site. The plasmid isthen cleaved with BamHI. The vector is cleaved with HindIII, partiallyfilled in and cleaved with BamHI. This enables insertion of the LHYpromoter fragment into the vector such that it drives luciferaseexpression. This construct is introduced into Agrobacterium, and thesebacteria used to introduce the T-DNA into Arabidopsis by selection ofkanamycin resistance. In the transgenic plants expression of luciferasemay be used to show the promoter fragment driving circadianrhythm-regulated gene expression as has been shown previously for thepromoter of the CAB2 gene of Arabidopsis (Millar et al, 1992).

The promoter may be used to drive other gene products in this way. Thisenables gene products of interest to be expressed around dawn. This isan advantage if plant growth and development is disrupted by the geneproduct when it is constantly expressed, for example because it isharmful to the cell during extended exposure to light or darkness. Italso enables the gene product to interact with gene products present atthe same time during the circadian cycle, and not to interact with thosepresent at different times during the cycle.

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TABLES

Flowering times and growth rates of lhy mutant and wild-type plants.Flowering times are shown as leaf numbers at flowering, and weremeasured in two experiments independently. The Table describingExperiment 2 illustrates that the mutation is dominant; plantsheterozygous for the lhy mutation flower at the same time as those thatare homozygous for the mutation. Growth rate is shown as number ofleaves formed each day, and is significantly slower for lhy than forwild-type plants grown under short days.

TABLE 1 Flowering time of lhy mutants under long and short days.Genotype No. and Daylength No. rosette leaves cauline leaves Total no.leaves A. Number of rosette and cauline leaves formed by lhy mutantsunder long and short days. lhy (LD) 12.0 (+/−0.5) 5.0 (+/−0.3) 17.0(+/−0.7) lhy (SD) 14.6 (+/−0.4) 4.3 (+/−0.2) 18.9 (+/−0.4) Landsbergerecta  4.6 (+/−0.2) 2.8 (+/−0.1) 7.4 (+/−0.2) (LD) Landsberg erecta27.4 (+/−0.5) 9.7 (+/−0.3) 37.0 (+/−0.5) (SD) B. Flowering time of lhymutants. Genotype Time to flowering Total no. leaves at and Daylength(Days) flowering lhy (LD) 33.2 (+/−1.8) 13.5 (+/−1.9) lhy (SD) 36.8(+/−1.7) 11.2 (+/−1.2) Landsberg erecta (LD) 20.1 (+/−1.5) 6.6 (+/−0.7)Landsberg erecta (SD) 35.6 (+/−1.7) 19.6 (+/−2.2)

TABLE 2 Rate of leaf formation of lhy and wild type. lhy wt ConditionsRate lves/day LR Rate lves/day LR Long days 0.85 0.997 Short days 0.520.995 1.05 0.999

27 1 2526 DNA Arabidopsis thaliana CDS (338)..(2275) 1 cagttatcttcttccttctt ctctctgttt tttaaattta tttttagaga attttttttg 60 ttttgcttccgatttgatta tttccgggaa cgatgacttc tccggggagt tcccggtgag 120 atgataagtcagattgcata cttgtctcct ccatggctac tctcaagggt tttggctgcg 180 gtggattcgtttggtttctc tagaatctaa agaggttatc acaacggctt tgcaatttga 240 aaactttcatgtttggggag atcaaagatg gtttcttttt tatactttac ttgttagaga 300 ggatttgaagcagcgaatag ctgcaaccgg tcctgtt atg gat act aat aca tct 355 Met Asp ThrAsn Thr Ser 1 5 gga gaa gaa tta tta gct aag gca aga aag cca tat aca ataaca aag 403 Gly Glu Glu Leu Leu Ala Lys Ala Arg Lys Pro Tyr Thr Ile ThrLys 10 15 20 cag cga gag cga tgg act gag gat gag cat gag agg ttt cta gaagcc 451 Gln Arg Glu Arg Trp Thr Glu Asp Glu His Glu Arg Phe Leu Glu Ala25 30 35 ttg agg ctt tat gga aga gct tgg caa cga att gaa gaa cat att ggg499 Leu Arg Leu Tyr Gly Arg Ala Trp Gln Arg Ile Glu Glu His Ile Gly 4045 50 aca aag act gct gtt cag atc aga agt cat gca caa aag ttc ttc aca547 Thr Lys Thr Ala Val Gln Ile Arg Ser His Ala Gln Lys Phe Phe Thr 5560 65 70 aag ttg gag aaa gag gct gaa gtt aaa ggc atc cct gtt tgc caa gct595 Lys Leu Glu Lys Glu Ala Glu Val Lys Gly Ile Pro Val Cys Gln Ala 7580 85 ttg gac ata gaa att ccg cct cct cgt cct aaa cga aaa ccc aat act643 Leu Asp Ile Glu Ile Pro Pro Pro Arg Pro Lys Arg Lys Pro Asn Thr 9095 100 cct tat cct cga aaa cct ggg aac aac ggt aca tct tcc tct caa gta691 Pro Tyr Pro Arg Lys Pro Gly Asn Asn Gly Thr Ser Ser Ser Gln Val 105110 115 tca tca gca aaa gat gca aaa ctt gtt tca tcg gcc tct tct tca cag739 Ser Ser Ala Lys Asp Ala Lys Leu Val Ser Ser Ala Ser Ser Ser Gln 120125 130 ttg aat cag gcg ttc ttg gat ttg gaa aaa atg ccg ttc tct gag aaa787 Leu Asn Gln Ala Phe Leu Asp Leu Glu Lys Met Pro Phe Ser Glu Lys 135140 145 150 aca tca act gga aaa gaa aat caa gat gag aat tgc tcg ggt gtttct 835 Thr Ser Thr Gly Lys Glu Asn Gln Asp Glu Asn Cys Ser Gly Val Ser155 160 165 act gtg aac aag tat ccc tta cca acg aaa cag gta agt ggc gacatt 883 Thr Val Asn Lys Tyr Pro Leu Pro Thr Lys Gln Val Ser Gly Asp Ile170 175 180 gaa aca agt aag acc tca act gtg gac aac gcg gtt caa gat gttccc 931 Glu Thr Ser Lys Thr Ser Thr Val Asp Asn Ala Val Gln Asp Val Pro185 190 195 aag aag aac aaa gac aaa gat ggt aac gat ggt act act gtg cacagc 979 Lys Lys Asn Lys Asp Lys Asp Gly Asn Asp Gly Thr Thr Val His Ser200 205 210 atg caa aac tac cct tgg cat ttc cac gca gat att gtg aac gggaat 1027 Met Gln Asn Tyr Pro Trp His Phe His Ala Asp Ile Val Asn Gly Asn215 220 225 230 ata gca aaa tgc cct caa aat cat ccc tca ggt atg gta tctcaa gac 1075 Ile Ala Lys Cys Pro Gln Asn His Pro Ser Gly Met Val Ser GlnAsp 235 240 245 ttc atg ttt cat cct atg aga gaa gaa act cac ggg cac gcaaat ctt 1123 Phe Met Phe His Pro Met Arg Glu Glu Thr His Gly His Ala AsnLeu 250 255 260 caa gct aca aca gca tct gct act act aca gct tct cat caagcg ttt 1171 Gln Ala Thr Thr Ala Ser Ala Thr Thr Thr Ala Ser His Gln AlaPhe 265 270 275 cca gct tgt cat tca cag gat gat tac cgt tcg ttt ctc cagata tca 1219 Pro Ala Cys His Ser Gln Asp Asp Tyr Arg Ser Phe Leu Gln IleSer 280 285 290 tct act ttc tcc aat ctt att atg tca act ctc cta cag aatcct gca 1267 Ser Thr Phe Ser Asn Leu Ile Met Ser Thr Leu Leu Gln Asn ProAla 295 300 305 310 gct cat gct gca gct aca ttc gct gct tcg gtc tgg ccttat gcg agt 1315 Ala His Ala Ala Ala Thr Phe Ala Ala Ser Val Trp Pro TyrAla Ser 315 320 325 gtc ggg aat tct ggt gat tca tca acc cca atg agc tcttct cct cca 1363 Val Gly Asn Ser Gly Asp Ser Ser Thr Pro Met Ser Ser SerPro Pro 330 335 340 agt ata act gcc att gcc gct gct aca gta gct gct gcaact gct tgg 1411 Ser Ile Thr Ala Ile Ala Ala Ala Thr Val Ala Ala Ala ThrAla Trp 345 350 355 tgg gct tct cat gga ctt ctt cct gta tgc gct cca gctcca ata aca 1459 Trp Ala Ser His Gly Leu Leu Pro Val Cys Ala Pro Ala ProIle Thr 360 365 370 tgt gtt cca ttc tca act gtt gca gtt cca act cca gcaatg act gaa 1507 Cys Val Pro Phe Ser Thr Val Ala Val Pro Thr Pro Ala MetThr Glu 375 380 385 390 atg gat acc gtt gaa aat act caa ccg ttt gag aaacaa aac aca gct 1555 Met Asp Thr Val Glu Asn Thr Gln Pro Phe Glu Lys GlnAsn Thr Ala 395 400 405 ctg caa gat caa acc ttg gct tcg aaa tct cca gcttca tca tct gat 1603 Leu Gln Asp Gln Thr Leu Ala Ser Lys Ser Pro Ala SerSer Ser Asp 410 415 420 gat tca gat gag act gga gta acc aag cta aat gccgac tca aaa acc 1651 Asp Ser Asp Glu Thr Gly Val Thr Lys Leu Asn Ala AspSer Lys Thr 425 430 435 aat gat gat aaa att gag gag gtt gtt gtt act gccgct gtg cat gac 1699 Asn Asp Asp Lys Ile Glu Glu Val Val Val Thr Ala AlaVal His Asp 440 445 450 tca aac act gcc cag aag aaa aat ctt gtg gac cgctca tcg tgt ggc 1747 Ser Asn Thr Ala Gln Lys Lys Asn Leu Val Asp Arg SerSer Cys Gly 455 460 465 470 tca aat aca cct tca ggg agt gac gca gaa actgat gca tta gat aaa 1795 Ser Asn Thr Pro Ser Gly Ser Asp Ala Glu Thr AspAla Leu Asp Lys 475 480 485 atg gag aaa gat aaa gag gat gtg aag gag acagat gag aat cag cca 1843 Met Glu Lys Asp Lys Glu Asp Val Lys Glu Thr AspGlu Asn Gln Pro 490 495 500 gat gtt att gag tta aat aac cgt aag att aaaatg aga gac aac aac 1891 Asp Val Ile Glu Leu Asn Asn Arg Lys Ile Lys MetArg Asp Asn Asn 505 510 515 agc aac aac aat gca act act gat tcg tgg aaggaa gtc tcc gaa gag 1939 Ser Asn Asn Asn Ala Thr Thr Asp Ser Trp Lys GluVal Ser Glu Glu 520 525 530 ggt cgt ata gcg ttt cag gct ctc ttt gca agagaa aga ttg cct caa 1987 Gly Arg Ile Ala Phe Gln Ala Leu Phe Ala Arg GluArg Leu Pro Gln 535 540 545 550 agc ttt tcg cct cct caa gtg gca gag aatgtg aat aga aaa caa agt 2035 Ser Phe Ser Pro Pro Gln Val Ala Glu Asn ValAsn Arg Lys Gln Ser 555 560 565 gac acg tca atg cca ttg gct cct aat ttcaaa agc cag gat tct tgt 2083 Asp Thr Ser Met Pro Leu Ala Pro Asn Phe LysSer Gln Asp Ser Cys 570 575 580 gct gca gac caa gaa gga gta gta atg atcggt gtt gga aca tgc aag 2131 Ala Ala Asp Gln Glu Gly Val Val Met Ile GlyVal Gly Thr Cys Lys 585 590 595 agt ctt aaa acg aga cag aca gga ttt aagcca tac aag aga tgt tca 2179 Ser Leu Lys Thr Arg Gln Thr Gly Phe Lys ProTyr Lys Arg Cys Ser 600 605 610 atg gaa gtg aaa gag agc caa gtt ggg aacata aac aat caa agt gat 2227 Met Glu Val Lys Glu Ser Gln Val Gly Asn IleAsn Asn Gln Ser Asp 615 620 625 630 gaa aaa gtc tgc aaa agg ctt cga ttggaa gga gaa gct tct aca tga 2275 Glu Lys Val Cys Lys Arg Leu Arg Leu GluGly Glu Ala Ser Thr 635 640 645 cagacttgga ggtaaaaaaa aaacatccacatttttatca atatctttaa atctagtgtt 2335 agtagtttgc ttctccaatc tttatgaaagagacttttaa ttttccttcc gaacatttct 2395 ttggtcatgt caggttctgt accatattaccccatgtctt gtctcttgtc tctgtttgtg 2455 tatgctactt gtggtctata tgtcatctgctactactgtt aattaaccat taagcaatgg 2515 atttgtcttt a 2526 2 645 PRTArabidopsis thaliana 2 Met Asp Thr Asn Thr Ser Gly Glu Glu Leu Leu AlaLys Ala Arg Lys 1 5 10 15 Pro Tyr Thr Ile Thr Lys Gln Arg Glu Arg TrpThr Glu Asp Glu His 20 25 30 Glu Arg Phe Leu Glu Ala Leu Arg Leu Tyr GlyArg Ala Trp Gln Arg 35 40 45 Ile Glu Glu His Ile Gly Thr Lys Thr Ala ValGln Ile Arg Ser His 50 55 60 Ala Gln Lys Phe Phe Thr Lys Leu Glu Lys GluAla Glu Val Lys Gly 65 70 75 80 Ile Pro Val Cys Gln Ala Leu Asp Ile GluIle Pro Pro Pro Arg Pro 85 90 95 Lys Arg Lys Pro Asn Thr Pro Tyr Pro ArgLys Pro Gly Asn Asn Gly 100 105 110 Thr Ser Ser Ser Gln Val Ser Ser AlaLys Asp Ala Lys Leu Val Ser 115 120 125 Ser Ala Ser Ser Ser Gln Leu AsnGln Ala Phe Leu Asp Leu Glu Lys 130 135 140 Met Pro Phe Ser Glu Lys ThrSer Thr Gly Lys Glu Asn Gln Asp Glu 145 150 155 160 Asn Cys Ser Gly ValSer Thr Val Asn Lys Tyr Pro Leu Pro Thr Lys 165 170 175 Gln Val Ser GlyAsp Ile Glu Thr Ser Lys Thr Ser Thr Val Asp Asn 180 185 190 Ala Val GlnAsp Val Pro Lys Lys Asn Lys Asp Lys Asp Gly Asn Asp 195 200 205 Gly ThrThr Val His Ser Met Gln Asn Tyr Pro Trp His Phe His Ala 210 215 220 AspIle Val Asn Gly Asn Ile Ala Lys Cys Pro Gln Asn His Pro Ser 225 230 235240 Gly Met Val Ser Gln Asp Phe Met Phe His Pro Met Arg Glu Glu Thr 245250 255 His Gly His Ala Asn Leu Gln Ala Thr Thr Ala Ser Ala Thr Thr Thr260 265 270 Ala Ser His Gln Ala Phe Pro Ala Cys His Ser Gln Asp Asp TyrArg 275 280 285 Ser Phe Leu Gln Ile Ser Ser Thr Phe Ser Asn Leu Ile MetSer Thr 290 295 300 Leu Leu Gln Asn Pro Ala Ala His Ala Ala Ala Thr PheAla Ala Ser 305 310 315 320 Val Trp Pro Tyr Ala Ser Val Gly Asn Ser GlyAsp Ser Ser Thr Pro 325 330 335 Met Ser Ser Ser Pro Pro Ser Ile Thr AlaIle Ala Ala Ala Thr Val 340 345 350 Ala Ala Ala Thr Ala Trp Trp Ala SerHis Gly Leu Leu Pro Val Cys 355 360 365 Ala Pro Ala Pro Ile Thr Cys ValPro Phe Ser Thr Val Ala Val Pro 370 375 380 Thr Pro Ala Met Thr Glu MetAsp Thr Val Glu Asn Thr Gln Pro Phe 385 390 395 400 Glu Lys Gln Asn ThrAla Leu Gln Asp Gln Thr Leu Ala Ser Lys Ser 405 410 415 Pro Ala Ser SerSer Asp Asp Ser Asp Glu Thr Gly Val Thr Lys Leu 420 425 430 Asn Ala AspSer Lys Thr Asn Asp Asp Lys Ile Glu Glu Val Val Val 435 440 445 Thr AlaAla Val His Asp Ser Asn Thr Ala Gln Lys Lys Asn Leu Val 450 455 460 AspArg Ser Ser Cys Gly Ser Asn Thr Pro Ser Gly Ser Asp Ala Glu 465 470 475480 Thr Asp Ala Leu Asp Lys Met Glu Lys Asp Lys Glu Asp Val Lys Glu 485490 495 Thr Asp Glu Asn Gln Pro Asp Val Ile Glu Leu Asn Asn Arg Lys Ile500 505 510 Lys Met Arg Asp Asn Asn Ser Asn Asn Asn Ala Thr Thr Asp SerTrp 515 520 525 Lys Glu Val Ser Glu Glu Gly Arg Ile Ala Phe Gln Ala LeuPhe Ala 530 535 540 Arg Glu Arg Leu Pro Gln Ser Phe Ser Pro Pro Gln ValAla Glu Asn 545 550 555 560 Val Asn Arg Lys Gln Ser Asp Thr Ser Met ProLeu Ala Pro Asn Phe 565 570 575 Lys Ser Gln Asp Ser Cys Ala Ala Asp GlnGlu Gly Val Val Met Ile 580 585 590 Gly Val Gly Thr Cys Lys Ser Leu LysThr Arg Gln Thr Gly Phe Lys 595 600 605 Pro Tyr Lys Arg Cys Ser Met GluVal Lys Glu Ser Gln Val Gly Asn 610 615 620 Ile Asn Asn Gln Ser Asp GluLys Val Cys Lys Arg Leu Arg Leu Glu 625 630 635 640 Gly Glu Ala Ser Thr645 3 18 DNA Artificial Sequence Description of Artificial SequencePrimer 3 atggatacta atacatct 18 4 18 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 4 ctagatttaa agatatta 18 5 125 DNAArtificial Sequence Description of Artificial Sequence Mutant 5cgttaccgac cgttttcatc cctatactca aaagagtaac cagtacgttt gattcgtctt 60gatggaactc aaagctaagt attttcaaat tacattgtgg atgatccaga tgtgagcaag 120tgatt 125 6 666 DNA Artificial Sequence Description of ArtificialSequence Mutant 6 atcctacttt catccctgct aaagaggtta tcacaacggc tttgcaatttgaaaactttc 60 atgtttgggg agatcaaaga tggtttcttt tttatacttt acttgttagagaggatttga 120 agcagcgaat agctgcaccg gtcctgttat ggatactaat acatctggagaagaattatt 180 agctaaggta ctactactaa tgaaataaga ttggtgtttt tttgtttgagagatttggac 240 tgttgttgtg tgaagatttg attttctttt gggttttcaa atgtttaggcaagaaagcca 300 tatacaataa caaagcagcg agrgcgatgg actgaggatg agcatgagaggtttctagaa 360 gccttgaggc tttatggaag agcttggcaa cgaattgaag gtcgraaggtttatcttttg 420 aatgtttagt ttgaactctt tgagatttta tattcctttg tttaggagtgtctttatctc 480 ctcttgattg ggagattcct tcttttcttt tcattttgtg tgcagaacatattgggacaa 540 agactgctgt tcagatcaga agtcatgcac aaaagttctt cacaaaggtaagttgatgat 600 cctttcagat cccggtgaaa cggtcgggaa actagctcta ccgtttccgtttccgtttac 660 cgtttt 666 7 55 PRT Arabidopsis thaliana 7 Thr Ile ThrLys Gln Arg Glu Arg Trp Thr Glu Asp Glu His Glu Arg 1 5 10 15 Phe LeuGlu Ala Leu Arg Leu Tyr Gly Arg Ala Trp Gln Arg Ile Glu 20 25 30 Glu HisIle Gly Thr Lys Thr Ala Val Gln Ile Arg Ser His Ala Gln 35 40 45 Lys PhePhe Thr Lys Leu Glu 50 55 8 16 PRT Unknown Organism Description ofUnknown Organism TEA domain of TEC1 (Swissprot database) 8 Trp Ser GluLys Val Glu Glu Ala Phe Leu Glu Ala Leu Arg Leu Ile 1 5 10 15 9 16 PRTUnknown Organism Description of Unknown Organism TEA domain of TEF1(Swissprot database) 9 Trp Ser Pro Asp Ile Glu Gln Ser Phe Gln Glu AlaLeu Ala Ile Tyr 1 5 10 15 10 41 PRT Unknown Organism Description ofUnknown Organism Accesion no. P25357 10 Trp Ser Val Arg Glu Ser Gln LeuPhe Pro Glu Leu Leu Lys Glu Phe 1 5 10 15 Gly Ser Gln Trp Ser Leu IleSer Glu Lys Leu Gly Thr Lys Ser Thr 20 25 30 Thr Asn Val Arg Asn Tyr TyrGln Arg 35 40 11 47 PRT Unknown Organism Description of Unknown OrganismBAS1 R1 (Swissprot Accession no. P22035) 11 Trp Thr Gln Glu Glu Asp GluGln Leu Leu Lys Ala Tyr Glu Glu His 1 5 10 15 Gly Pro His Trp Leu SerIle Ser Met Asp Ile Pro Gly Arg Thr Glu 20 25 30 Asp Gln Cys Ala Lys ArgTyr Ile Glu Val Leu Gly Pro Gly Ser 35 40 45 12 47 PRT Unknown OrganismDescription of Unknown Organism BAS1 R2 (Swissprot Accession no. P22035)12 Trp Thr Leu Glu Glu Asp Leu Asn Leu Ile Ser Lys Val Lys Ala Tyr 1 510 15 Gly Thr Lys Trp Arg Lys Ile Ser Ser Glu Met Glu Phe Arg Pro Ser 2025 30 Leu Thr Cys Arg Asn Arg Trp Arg Lys Ile Ile Thr Met Val Val 35 4045 13 57 PRT Arabidopsis thaliana 13 Thr Ile Thr Lys Gln Arg Glu Arg TrpThr Glu Asp Glu His Glu Arg 1 5 10 15 Phe Leu Glu Ala Leu Arg Leu TyrGly Arg Ala Trp Gln Arg Ile Glu 20 25 30 Glu His Ile Gly Thr Lys Thr AlaVal Gln Ile Arg Ser His Ala Gln 35 40 45 Lys Phe Phe Thr Lys Leu Glu LysAla 50 55 14 83 PRT Arabidopsis thaliana 14 Met Asp Thr Asn Thr Ser GlyGlu Glu Leu Leu Ala Lys Ala Arg Lys 1 5 10 15 Pro Tyr Thr Ile Thr LysGln Arg Glu Arg Trp Thr Glu Asp Glu His 20 25 30 Glu Arg Phe Leu Glu AlaLeu Arg Leu Tyr Gly Arg Ala Trp Gln Arg 35 40 45 Ile Glu Glu His Ile GlyThr Lys Thr Ala Val Gln Ile Arg Ser His 50 55 60 Ala Gln Lys Phe Phe ThrLys Leu Glu Lys Glu Ala Glu Val Lys Gly 65 70 75 80 Ile Pro Val 15 111PRT Arabidopsis thaliana 15 Gly Arg Ile Ala Phe Gln Ala Leu Phe Ala ArgGlu Arg Leu Pro Gln 1 5 10 15 Ser Phe Ser Pro Pro Gln Val Ala Glu AsnVal Asn Arg Lys Gln Ser 20 25 30 Asp Thr Ser Met Pro Leu Ala Pro Asn PheLys Ser Gln Asp Ser Cys 35 40 45 Ala Ala Asp Gln Glu Gly Val Val Met IleGly Val Gly Thr Cys Lys 50 55 60 Ser Leu Lys Thr Arg Gln Thr Gly Phe LysPro Tyr Lys Arg Cys Ser 65 70 75 80 Met Glu Val Lys Glu Ser Gln Val GlyAsn Ile Asn Asn Gln Ser Asp 85 90 95 Glu Lys Val Cys Lys Arg Leu Arg LeuGlu Gly Glu Ala Ser Thr 100 105 110 16 83 PRT Arabidopsis thaliana 16Ile Ala Thr Thr Glu Ala Gly Glu Ala Pro Glu Lys Lys Val Arg Lys 1 5 1015 Ala Tyr Thr Ile Thr Lys Ser Arg Glu Ser Trp Thr Glu Gly Glu His 20 2530 Asp Lys Phe Leu Glu Ala Leu Gln Leu Phe Asp Arg Asp Trp Lys Lys 35 4045 Ile Glu Asp Phe Phe Gly Ser Lys Thr Val Ile Gln Ile Arg Ser His 50 5560 Ala Gln Lys Tyr Phe Leu Lys Val Gln Lys Asn Gly Thr Leu Ala His 65 7075 80 Ile Pro Thr 17 118 PRT Oryza sativa 17 Phe Asp Ala Leu Phe Ser ArgGlu Arg Leu Pro Gln Ser Phe Ser Pro 1 5 10 15 Pro Gln Val Glu Gly SerLys Glu Ile Ser Lys Glu Glu Glu Asp Glu 20 25 30 Val Thr Thr Val Thr ValAsp Leu Asn Lys Asn Ala Ala Ile Ile Asp 35 40 45 Gln Glu Leu Asp Thr AlaAsp Glu Pro Arg Ala Ser Phe Pro Asn Glu 50 55 60 Leu Ser Asn Leu Lys LeuLys Ser Arg Arg Thr Gly Phe Lys Pro Tyr 65 70 75 80 Lys Arg Cys Ser ValGlu Ala Lys Glu Asn Arg Val Pro Ala Ser Asp 85 90 95 Glu Val Gly Thr LysArg Ile Arg Leu Glu Ser Glu Asp Arg His Asp 100 105 110 Leu Leu Ser ThrTrp Val 115 18 1738 DNA Arabidopsis thaliana 18 caaacatcaa cgtagggatccgtgaaatat ttaaatccgg tttgtttggt tattttggaa 60 taatttcggt tatttcaattagattcgggt agttcagttc ttcggttagt aacaaaaact 120 ggtctattgt tttttggttaacctagaacc gaaccgaact aaccaaagtt ctcggtaacc 180 ttttgtagtg gcttcctgaccgatgaggcc gtcaacttca aaaaatattg caactaagct 240 ctgctccaaa attagagtatctataactat gttaaacgct tctgctttaa gcaaaacaca 300 gttgtaagct ggaatctaaaaaaatgagtg taatgatgtt tgctgaattc cataaataaa 360 tactacatgc ttcggttaagacttaagagt aattaatgtt ccttaatttc tacaaatgtt 420 atataagcaa gttgaccaaagttctcgatg ataatttgtt gaaattttgt ataggcattg 480 catgatatta tatgaaaagatgaagatttt tatacagacg caagttcccc gagcagtcca 540 agcttgtcgg gtttaattcaactatgttaa tacgcaaatt tatatagaat aggcgtaaaa 600 gtgaggccca tacaatgtcttattacaagc ccagatccag catagccaat acgtagcagt 660 acaccatcac agctggcaccgtacccactt gtttagtcgt ccaagtttgt accaataatc 720 gtttacacgt aagcaattgtggaccaccac actcactttt acctacgtga gcttcacatt 780 gaagcttctg gctcgtagagaagcaacttg agatatacca aaaagtgcag tagacagcca 840 ctacaatatc accacgtgtcgatctgcgat gacttctgtt tttccattta tacccttggt 900 gctgttccag cctcaaataacttttcaatt aaaatttttc caaaaattag gggcaaaaat 960 tgttgtggct gagattgcttctggcttctc ttcttcttct tccagtcttc ttcagcctaa 1020 aacagtcttc cttcttcttcttcttcttct tcttctttca gttatcttct tccttcttct 1080 ctctgttttt taaatttatttttagagatt tttttttgtt ttgcttccga tttgattatt 1140 tccgggaacg atgacttctccggggagttc ccggtgagat gataagtcag attgcatact 1200 tgtctcctcc atggctactctcaagggtat aacagtttac attatgagca gtttctagga 1260 ttcctataac atactaagatctctgtttgg ctgctgagaa acttatacaa gcgcattaac 1320 taaatcttat tagctctaaaagttagcata aatgatacga atctggtgat tgattactga 1380 tatgaagatt tgtgaaggttttggctgagg tggattcgtt tgggtgaggc ttttgtgaat 1440 aataataaag ggaattcttttgagttctgc tggagaagca gcgactgttt cacggtggtc 1500 tttgaaaaga tttctcttttgaatttcgct catcactctt atcttagtgt ttgtggataa 1560 atatttctca taaagtactttctcctttgc agtttctcta gaatctaaag aggttatcac 1620 aacggctttg caatttgaaaactttcatgt ttggggagat caaagatggt ttctttttta 1680 tactttactt gttagagaggatttgaagca gcgaatagct gcaaccggtc ctgttatg 1738 19 447 DNA UnknownOrganism Description of Unknown Organism Nucleotide sequence of EST162I3T7, Accession no. R30439 19 ctagacgatc tctatcttga ataaaataccgataatnacc tcaaccaatc cggtggtcgc 60 cgaagtaata ccggcggaaa cttctacagatgctacagag acgacgattg caacgacgga 120 agctggtgaa gcaccggaga agaaggtgaggaaagcttac acaatcacca agtctagaga 180 gagttggact gaaggagaac acgncaagtttctggaagct cttcaattgt ttgatcgtga 240 ctggaaaaag atagaagatt tttttggttcaaagncagtt attcagatca ggagccatgc 300 ccagaaatac tttctaaagg tccaaaaaaatgggncttta gcacatnttc ccncccccta 360 ggnctanggg caaagtngct catncatatccnnaaaaggc attcgaaaaa ttgctcaant 420 ttcggttnnc gtttcnatng cctttcc 44720 26 DNA Artificial Sequence Description of Artificial Sequence Primer20 gatataccgg taacgaaaac gaacgg 26 21 26 DNA Artificial SequenceDescription of Artificial Sequence Primer 21 ttcgtttccg tcccgcaagttaaata 26 22 25 DNA Artificial Sequence Description of ArtificialSequence Primer 22 cgttaccgac cgtttttcat cccta 25 23 24 DNA ArtificialSequence Description of Artificial Sequence Primer 23 acgaacgggataaatacggt aatc 24 24 26 DNA Artificial Sequence Description ofArtificial Sequence Primer 24 gttagtttta tcccgatcga tttcga 26 25 21 DNAArtificial Sequence Description of Artificial Sequence Primer 25accgctttga ttgagaagct g 21 26 26 DNA Artificial Sequence Description ofArtificial Sequence Primer 26 catcaacgta gggatccgtg aaatat 26 27 31 DNAArtificial Sequence Description of Artificial Sequence Primer 27ggactagtaa caggaccggt tgcagctatt c 31

What is claimed is:
 1. An isolated polynucleotide nucleic acid isolateencoding a polypeptide which comprises the amino acid sequence shown inFIG. 1 (SEQ ID NO: 2) and has the functional activity of the polypeptideof SEQ ID NO: 2, which functional activity is delaying flowering time.2. A polynucleotide according to claim 1 wherein the coding nucleotidesequence is the coding nucleotide sequence shown in FIG. 1 (SEQ IDNO:1).
 3. A polynucleotide according to claim 1 operably linked to aregulatory sequence for expression.
 4. An isolated polynucleotide whichcomprises a nucleotide sequence complementary to 300 contiguousnucleotides of the sequence of claim
 1. 5. A method of producing aplant, the method including incorporating a heterologous polynucleotideaccording to claim 4 into a plant cell and regenerating a plant fromsaid plant cell.
 6. A method of advancing flowering time of a plant, themethod including causing or allowing expression from a polynucleotideaccording to claim 4 within cells of the plant.
 7. A polynucleotideaccording to claim 1 operably linked to a regulatory sequence fortranscription.
 8. A polynucleotide according to claim 7 wherein theregulatory sequence includes an inducible promoter.
 9. A nucleic acidvector suitable for transformation of a plant or microbial host andincluding a polynucleotide according to claim
 1. 10. A plant ormicrobial cell containing a heterologous polynucleotide or nucleic acidvector according to claim
 9. 11. A plant including a cell according toclaim
 10. 12. A part or propagule of a plant including a cell accordingto claim
 10. 13. A method of producing a plant, the method includingincorporating a heterologous polynucleotide according to claim 1 into aplant cell and regenerating a plant from said plant cell.
 14. A methodaccording to claim 13 or claim 5 including sexually or asexuallypropagating or growing off-spring or a descendent of said plant, whereinsaid off-spring or descendant comprises said heterologouspolynucleotide.
 15. A method of delaying flowering time of a plant, themethod including causing or allowing expression of a product encoded bya heterologous polynucleotide according to claim 1 within cells of theplant.